MEIDICAL 


COLLECT  OF   PHARfciACY 
Dept.    of  Bacteriology 


PHARMACEUTICAL 
BACTERIOLOGY 


SCHNEIDER 


PHARMACEUTICAL 
BACTERIOLOGY 


BY 

ALBERT  SCHNEIDER,  M.  D.,  Ph.  D. 

(Columbia  University) 

PROFESSOR  OF  PHARMACOGNOSY,   COLLEGE  OF  PHARMACY; 
UNIVERSITY  OF  NEBRASKA,   LINCOLN. 


SECOND  EDITION  REVISED  AND  ENLARGED 
WITH  97  ILLUSTRATIONS 


PHILADELPHIA 

P.   BLAKISTON'S   SON   &   CO 

1012   WALNUT  STREET 
1920 


COPYRIGHT,  1920,  BY  P.  BLAKISTON'S  SON  &  Co. 


THE  MAPLE  PRESS  YORK  PA 


PREFACE  TO  SECOND  EDITION 


The  recent  progress  in  bacteriological  science  has  made  it  necessary 
to  make  certain  changes  and  additions  in  the  present  volume.  The  fol- 
lowing chapters  have  been  added.  Chapter  III,  The  Origin  of  Bacteria; 
Chapter  VII,  Symbiology;  Chapter  IX,  Zymology;  and  Chapter  XII, 
Adenology.  In  addition  to  this  wholly  new  matter,  not  given  in  the  first 
edition,  other  additions  have  been  made  in  the  text.  The  subject  of  soil 
bacteriology  and  of  milk  and  water  analysis,  has  been  treated  more  fully. 
The  chapter  on  sterilization  and  disinfection  has  been  enlarged.  A  brief 
statement  of  sensitized  bacterins,  of  anaphylaxis,  of  aggressins  and  of 
storing  biologies  has  been  introduced.  Some  of  the  disputed  points 
have  been  cleared  up  or  have  been  entirely  omitted.  The  many  imper- 
fections of  a  first  edition  of  a  book  covering  a  new  field  of  scientific  en- 
deavor, or  to  put  it  more  correctly,  a  new  application  of  a  science,  namely 
the  science  of  bacteriology  to  pharmacy,  have  been  largely  corrected. 

A  text-book  deals  with  the  established  facts  of  science,  giving  just 
enough  of  the  theoretical  to  indicate  the  further  advance  in  the  near  or 
perhaps  remote  future.  The  present  volume  has  adhered  to  this  require- 
ment. It  is  possible  to  present  the  facts  of  science  intelligently  without 
using  difficult  words  or  complicated  phraseology.  A  suitable  text  must 
not  be  encumbered  by  unnecessary  technical  terms,  nor  should  it  be  a 
mere  glossary  ^of  scientific  terms.  If  definitions  were  all  that  is  required 
of  a  text-book,  then  Webster's  Unabridged  Dictionary  would  be  the  ideal 
universal  text-book  for  all  grades  and  classes  of  students.  It  is  believed 
that  the  present  volume  is  not  faulty  in  these  regards.  It  is  believed  that 
the  book  will  be  found  quite  "readable"  and  intelligible  to  the 
student  of  ordinary  ability. 

Grateful  acknowledgments  are  hereby  made  to  Dr.  Aubrey  H. 
Straus  of  the  Medical  College  of  Virginia,  for  many  suggestions  and  for 
calling  attention  to  some  of  the  more  glaring  imperfections  in  the  first 
edition.  The  present  volume  is  believed  to  be  complete  for  the  purpose 
for  which  it  is  intended. 


LINCOLN,  NEBRASKA, 
December,  1919. 


•40384 


PREFACE  TO  FIRST  EDITION 


The  recent^growth  and  development  of  the  professional  side  of  phar- 
macy has  made  new  text-books  necessary.  The  present  volume  is  the 
product  of  such  progress. 

The  illustrations  have  been  selected  with  a  view  to  a  fuller  explanation 
of  the  text.  The  descriptions  of  the  illustrations  have  been  made  unusu- 
ally complete.  This  is  to  make  it  possible  for  the  student  to  ascertain  the 
use  of  every  article  illustrated  without  the  necessity  of  searching  for  addi- 
tional information  in  the  text  itself.  Some  of  the  illustrations  are  from 
original  drawings,  others  are  from  electros  supplied  by  the  Bausch  & 
Lomb  Optical  Company  and  the  Cutter  Biological  Laboratory  of  Berk- 
eley, California.  Still  others  are  taken  from  recent  works  on  bacteriology, 
notably  Williams'  " Manual  of  Bacteriology." 

Attempts  have  been  made  to  adhere  strictly  to  the  subject  from  the 
standpoint  of  the  pharmacist,  with  only  enough  treatment  of  general 
bacteriology  to  make  clear  the  collateral  relationships,  especially  as  it 
pertains  to  medical,  and  commercial  or  industrial  bacteriology. 

While  this  volume  is  primarily  intended  for  students  in  colleges  of 
pharmacy,  it  is  hoped  it  will  also  be  found  useful  by  practising  pharmacists. 

SAN  FRANCISCO. 


' 


CONTENTS 


PAGE 

CHAPTER  I.— GENERAL  INTRODUCTION      I 

Introduction  of  bacteriology  into  colleges  of  pharmacy.  Relationship  of 
pharmaceutical  and  medical  bacteriology.  Reasons  why  pharmacists  should 
study  bacteriology. 

CHAPTER  II.— HISTORICAL 5 

Introduction.  From  Hippocrates  (300  B.  C.)  to  Leeuwenhoek  (1656),  the 
earliest  ideas  regarding  infections,  epidemics  and  spontaneous  generation. 
From  Leeuwenhoek  (1656)  to  Schwann  (1837),  the  discovery  of  micro-organ- 
isms and  the  earliest  observations  regarding  their  activities.  From  Schwann 
(1837)  to  Pasteur  (1862),  the  earlier  investigations  pertaining  to  the  relation- 
ship of  micro-organisms  to  fermentation  and  to  disease.  From  Pasteur  (1862) 
to  Behring  (1890),  period  of  remarkable  activity  in  bacteriological  pathology, 
listerism,  antiseptic  surgery,  etc.  From  Behring  (1890)  to  Wright  (1907),  dis- 
covery of  serum  therapy,  bacterial  vaccines  and  development  of  utilitarian 
bacteriology. 

CHAPTER  III. — THE  ORIGIN  OF  BACTERIA  AND  OF  OTHER  MICRO-ORGANISMS  .    .     22 
Spontaneous  generation.     The  vitalistic  hypothesis.     The  panspermia  theory. 
The  theory  of  universal  evolution.     The  colloid  theory.     Other  theories. 

CHAPTER  IV. — GENERAL  MORPHOLOGY  AND  PHYSIOLOGY  OF  BACTERIA      ...     35 
Classification  of  microbes.     General  morphology.     General  physiology. 

CHAPTER  V. — RANGE  AND  DISTRIBUTION  OF  BACTERIA 60 

Bacteria  of  earth,  air  and  water.  Bacteria  found  in  animals,  in  plants,  on  non- 
living objects,  etc.  Altitudinal  range.  Latitudinal  range. 

CHAPTER  VI. — BACTERIOLOGICAL  TECHNIC      63 

Cleaning  glassware.  Plugging  containers  with  cotton.  Filling  test-tubes  with 
culture  media.  Preparation  of  culture  media.  Sterilization  of  culture  media. 
Titrating  for  reaction  of  media.  Making  bacterial  cultures.  Making  bac- 
terial counts.  Preparing  bacterial  stains.  Examining  bacteria. 

CHAPTER  VII.— SYMBIOLOGY 119 

Definitions.  General  introduction  to  the  phenomena  of  symbiosis.  Forms 
of  symbiosis — accidental,  contingent  parasitism,  [commensalism,  nutricism, 
mutualism,  individualism,  paracytosis,  patrocytosis,  leucocytosis,  compound 
symbioses. 

CHAPTER  VIIL— BACTERIA  IN  THE  INDUSTRIES 158 

The  function  of  bacteria  in  agriculture.  Bacteria  in  milk  and  in  the  dairying 
industry.  Bacteria  in  water  supplies.  Bacterial  pest  exterminators.  Bac 
teria  in  the  tanning  industry.  Rotting  bacteria.  Cider  making. 

ix 


X  CONTENTS 

PAGE 

CHAPTER  IX. — ZYMOLOGY — FERMENTS  AND  FERMENTATION 210 

Introduction.  General  properties  of  enzymes.  Classification  of  ferments. 
A  brief  statement  of  the  properties  of  the  more  important  ferments. 

CHAPTER  X. — IMMUNOLOGY,  BACTERIAL  ACTIVITIES  AND  BACTERIAL  PRODUCTS  235 
Immunity,  natural  and  acquired.     Race,  age  and  sex  immunity.     Anaphy- 
laxis.     Phagocytosis.     Ehrlich's  side-chain  theory.     Toxins  and  antitoxins. 
Agglutinins.     Precipitins.    Lysins.     Opsonins  and  the  opsonic  index. 

CHAPTER  XI. — SEROLOGY.    THE    MANUFACTURE    AND    USE    OF    SERA    AND 

VACCINES ' ,,   258 

Antidiphtheric  serum.  Other  sera.  Bacterial  vaccines  (bacterins).  Concen- 
trat  ed  diphtheria  antitoxin .  M  anuf  act  ure  of  bacterial  vaccines .  Tuberculins . 
Small-pox  vaccine.  Rabies  vaccine. 

CHAPTER  XII. — ADENOLOGY— THE  GLANDS.     GLANDULAR  EXTRACTS 277 

The  glands  in^  (general.  The  ductless  glands.  The  interrelationship  of  the 
functional  activities  of  the  glands  and  of  the  body  cells.  The  ductless  glands 
in  health  and  in  disease.  Glandular  extracts  and  their  therapeutic  use. 

CHAPTER  XIII. — YEASTS  AND  MOULDS.  . 294 

Yeast  organisms.  Moulds.  Organisms  active  in  fermented  drinks,  as  beer, 
wine  and  sake.  •  Parasitic  yeasts.  Yeast  cakes. 

CHAPTER  XIV— PROTOZOA  IN  DISEASE 316 

Rhizopoda.     Flagellata.     Sporozoa. 

CHAPTER  XV. — DISINFECTANTS  AND   DISINFECTION.     FOOD   PRESERVATIVES. 

INSECTICIDES 321 

Principles  of  sterilization  and  disinfection.  Pasteurization.  Standardization 
of  disinfectants.  Methods  of  sterilization.  Disinfectants  and  their  use. 
Disinfection  of  rooms  and  buildings.  Disinfection  of  sewage.  Sterilization  of 
water  supplies.  Food  preservatives,  their  use  and  abuse.  Insecticides  and 
other  pest  exterminators. 

CHAPTER  XVI. — STERILIZATION  AND  DISINFECTION  IN  THE  PHARMACY  .'  .    .  357 
Sterilization     of    containers,     stoppers,     etc.     Surgical     supplies.     Dusting 
powders.     Salves  and  pastes.     Gargles,  washes,  etc.     Chemicals  and  galen- 
icals.    Solutions  for  hypodermic  use.     Ampuls. 

CHAPTER  XVII. — COMMUNICABLE  DISEASES  WITH  SUGGESTIONS  ON  PREVEN- 
TIVE MEDICINE 369 

Causes  of  disease.  Tuberculosis.  Typhoid  fever.  Pneumonia.  Small-pox. 
Malaria.  Diphtheria.  Cancer.  Plague.  Asiatic  cholera.  Yellow  fever. 
Pellagra.  Syphilis.  Gonorrhea.  Tabulation  of  diseases.  Disease  carriers. 

CHAPTER  XVIII.— A  BACTERIOLOGICAL  AND  MICROSCOPICAL  LABORATORY  FOR 

THE  PHARMACIST 400 

Necessary  qualifications  to  do  bacteriological  and  microanalytical  work. 
Position  of  laboratory.  Size  of  laboratory.  Furnishings.  Equipment.  Ap- 
paratus. Reference  library.  Outline  of  microscopical  and  bacteriological 
work.  The  exhibit  cabinet. 

GENERAL  INDEX 431 


PHARMACEUTICAL  BACTERIOLOGY 


CHAPTER  I 
GENERAL  INTRODUCTION 

The  science  of  bacteriology  fs  not  new,  but  its  introduction  into 
pharmacy  is  of  comparatively  recent  date. 

About  1896  a  few  of  the  colleges  of  pharmacy  in  the  United  States  gave 
optional  courses  of  instruction  in  bacteriology.  At  the  present  time  nearly 
all  of  the  leading  colleges  of  pharmacy  give  instruction  in  bacteriology  and 
in  many  of  these  institutions  the  courses  are  compulsory,  forming  a  part  of 
the  prescribed  curriculum,  represented  by  lectures  and  laboratory  work. 
In  some  universities  the  students  of  pharmacy  receive  their  bacteriological 
instruction  in  the  department  of  medicine  or  perhaps  dentistry.  However, 
pharmaceutical  bacteriology  and  medical  bacteriology  are  quite  distinct. 
Medical  students  study  this  subject  from  the  standpoint  of  pathology  and 
disease,  matters  which  concern  the  pharmacist  but  little.  Students  of 
pharmacy  do  not  have  the  time  necessary  to  devote  themselves  extensively 
to  special  laboratory  methods  and  technic,  nor  is  it  advisable  that  they 
should  receive  extensive  laboratory  instruction  in  pathology.  Pharma- 
ceutical bacteriology  must  be  suitably  adapted  to  the  practice  of  pharmacy. 

The  pharmacist  should  have  a  knowledge  of  general  bacteriology,  in 
order  that  he  may  realize  what  important  relationships  bacteria  bear  to 
human  activities  in  general,  to  medical  practice  more  especially,  and  in 
order  that  he  may  comprehend  quite  fully  the  significance  of  these  minute 
organisms  in  pharmaceutical  practice.  He  should  know  what  pharma- 
ceutical preparations  and  what  medicinal  substances  are  likely  to  be 
attacked  by  bacteria,  and  what  changes  they  are  capable  of  producing  in 
such  substances.  He  should  have  some  knowledge  of  the  effects  that 
bacterially  deteriorated  substances  may  have  when  introduced  into  the 
human  organism.  He  should  be  qualified  to  sterilize  pharmaceuticals  as 
is  now  required  in  the  U.  S.  P.  and  in  the  pharmacopoeias  of  several 
foreign  countries  as  Austria,  Italy,  and  Belgium.  He  should  know  some- 


2  PHARMACEUTICAL  BACTERIOLOGY 

thing  of  the  comparative  value  6f  the  numerous  disinfectants  and  anti- 
septics  used  and  found  upon   the  market   and  should  know  how   to 
standardize  these  agents  according  to  recent   bacteriological   methods. 
The  pharmacist  should  know  that  bacteria,  yeasts,  and  related  organisms 
develop  very  promptly  and  profusely  in  all  aromatic  waters;  in  carelessly 
manipulated  boiled  and  distilled  water;  in  dilute  solutions  of  all  acids  and 
alkalies;  in  dilute  alcohol  and  alcoholic  liquids;  tinctures;  infusions;  ex- 
tracts, solid  and  liquid;  decoctions;  in  dilute  salt  solutions;  in  plant  juices; 
mucilages;  emulsions;  elixirs;  wines;  in  syrups  of  all  kinds;  in  carelessly 
manipulated  vegetable  drugs,  crude  and  powdered;  in  drugs  from  the 
animal  kindgom,  as  ox-gall,  lard,  oils,  fats,  pepsin,  etc.     He  should  have  a 
clear  comprehension  of  antiseptics  as  germ  destroyers,  and  should  know 
how  to  prepare  and  use  them.     He  should  have  a  general  knowledge 
of  phagocytosis;  of  leucocytosis  in  inflammatory  processes,  in  pus  forma- 
tion, necrosis,  etc.     He  should  comprehend  immunity,  natural  and  ac- 
quired; he  should  know  about  opsonins  and  the  opsonic  index.     He  should 
have  a  general  knowledge  of  bacterial  enzymes;  of  toxins,  ptomaines, 
leucomaines;  of  antitoxins;  of  bacterial  vaccines.     He  should  have  a 
special  knowledge  of  the  source,  manufacture,  and  use  of  antitoxins  and 
toxins,  modified  toxins,  vaccine  virus,  and  related  products  used  in  med- 
ical practice.     He  should  have  a  general  knowledge  of  the  causation  of  the 
more  common  bacterial  and  protozoic  diseases.     He  should  have  special 
instruction  in  the  disinfection  of  public  and  private  dwellings,  and  should 
be  able  to  cooperate  with  the  physician  in  stamping  out  threatened  epi- 
demics and  in  carrying  out  public  prophylactic  and  hygienic  measures. 
To  attain  these  ends  a  knowledge  of  bacteriology,  specialized  to  suit  the 
needs  of  the  pharmacist,  is  absolutely  essential. 

It  is  not  the  so-called  practical  side  of  bacteriology,  represented  by 
dollars  and  cents,  which  should  interest  Jie  pharmacist  in  this  science,  but 
rather  the  broader  view  of  his  profession  which  will  enable  him  to  perform 
his  duties  more  intelligently  and  more  efficiently.  The  man  whose  actions 
are  altogether  prompted  and  directed  by  the  dollar  sign  has  no  place  in 
pharmacy  or  in  medicine.  He  should  turn  to  some  non-professional 
enterprise. 

Text-books  on  bacteriology  for  use  in  universities,  medical  colleges, 
and  technical  schools  are  not  suitable  for  use  in  colleges  of  pharmacy. 
Some  of  these  books  are  excellent  collateral  reading  for  pharmacists,  but 
most  of  them  are  of  such  a  highly  specialized  nature  that  they  would  no 
doubt  do  more  harm  than  good  should  the  average  pharmacist  attempt  to 
use  them  as  a  practical  guide  in  the  performance  of  his  duties.  Bacteri- 
ology must  not  be  made  discouragingly  difficult  to  the  pharmacist,  in 
order  that  the  best  results  may  be  attained. 


GENERAL  INTRODUCTION  3 

Wherever  possible  the  college  instruction  in  pharmaceutical  bacteri- 
ology should  be  supplemented  by  visits  to  biological  laboratories  for  the 
manufacture  of  sera  and  bacterial  vaccines,  to  board  of  health  laboratories, 
quarantine  stations,  garbage  reduction  works,  etc.  Students  should  also 
be  assigned  special  reading.  Journals  and  special  treatises  on  bacteriology 
and  on  public  sanitation  should  be  consulted.  The  reports  on  bacterio- 
logical and  related  subjects  issued  from  time  to  time  by  the  United~States 
Public  Health  Service  are  of  special  interest. 

The  following  references  are  given  for  the  benefit  of  those  students  who 
may  desire  further  information  regarding  the  earlier  conceptions  of  phar- 
maceutical bacteriology.  It  will  be  found  that  the  opinions  advanced  by 
the  authors  cited  differ  considerably. 

1.  Bacteriology   for   Pharmacists.    Pharm.  Journ.  Trans.,  23   (III), 
565,865;  24  (III),  101,  1893. 

Largely  a  description  of  the  apparatus  employed  in  bacteriological  work, 
giving  special  attention  to  the  value  and  use  of  the  compound  microscope 
in  such  work. 

2.  H.  P.  Campbell.     Bacteria  Dangerous  to  Medicines.     Am.  Journ. 
Pharm.,  72,  113-118,  1890. 

3.  R.  G.  Eccles.     Pharmaceutical  Bacteriology.    Proc.  A.  Ph.  A.,  42, 
225-230,  1894. 

A  very  interesting  paper  on  the  theoretical  possibilities  of  pharmaceuti- 
cal bacteriology. 

4.  J.  L.  Hatch.     Bacteriology.    Pharm.  Journ.  Trans.,  22  (III),  271, 
289,  330,  1891. 

A  series  of  lectures  delivered  before  the  alumni  association  of  the  Phila- 
delphia College  of  Pharmacy,  devoting  the  major  attention  to  the  morph- 
ology, physiology,  and  classification  of  bacteria. 

5.  R.  T.  Hewlett.     Bacteriology  in  its  Practical  Aspects.    Pharm. 
Journ.  Trans.,  25  (III),  819-820,  893-894,  1895. 

A  general  retrospect  of  bacteriology  as  a  possible  source  of  financial 
gain  to  the  pharmacist. 

6.  Smith  Ely  Jeliffe.     Moulds  and  Bacteria.     Druggists  Circular,  94— 
95.  1897. 

A  description  of  some  of  the  more  common  moulds  and  bacteria  found 
in  medicinal  solutions.  Good  illustrations. 

7.  E.  Klein.     Bacteria,  Their  Nature  and  Function.     Pharm.  Journ. 
Trans.,  23  (III),  15,  35,  1893. 

8.  W.    H.    Lymans.     Bacteriological    Culture    Apparatus.    Pharm. 
Journ.     Trans.,  1893.     (National  Druggist,  173,  1893.) 

9.  Albert  Schneider.     Pharmaceutical  Bacteriology.     Proc.  A.  Ph.  A., 
48,  186-189,  l894- 


4  PHARMACEUTICAL  BACTERIOLOGY 

10.  Specialism  in  Pharmacy,  begotten  by  Progress  in  Bacteriology. 
Pharm.  Journ.  Trans.,  25  (III),  625,  1895. 

Points  out  the  necessity  of  a  suitable  preparatory  training;  the  impor- 
tance of  a  knowledge  of  the  use  of  antiseptics. 

11.  R.   Warrington.     The   Chemistry  of  Bacteria.    Pharm.   Journ. 
Trans.,  23  (III),  402,  1893.     (Pharm.  Era.,  104,  1894.) 


CHAPTER  II 
HISTORICAL 

It  must  be  evident  that  the  science  of  bacteriology  had  its  inception 
with  the  discovery  of  the  compound  microscope.  For  some  time  the  progress 
in  bacteriological  investigation  continued  parallel  with  the  progress  in 
the  mechanical  perfection  of  the  microscope  and  with  the  advance  in 
microscopical  technic.  Gradually,  however,  the  chemical  and  physio- 
logical investigations  pertaining  to  bacteria  gained  in  importance  and 
significance.  Our  knowledge  of  the  morphology  of  bacteria  as  revealed 
through  the  compound  microscope  has  been  practically  stationary  for  two 
decades,  but  not  so  our  knowledge  of  bacterial  products  and  bacterial 
action.  The  methods  of  bacteriological  technology  have  been  gradually 
perfected,  and  the  progress  along  this  line  has  kept  pace  with  the  chemical 
and  physiological  investigations. 

Although,  as  indicated,  the  science  of  bacteriology  is  of  comparatively 
recent  origin,  yet  we  must  not  lose  sight  of  the  fact  that  many  of  the  ideas 
underlying  this  science,  as  now  comprehended,  were  advanced  in  remote 
antiquity.  For  this  reason  it  is  desirable  to  set  forth  these  earlier  concepts 
in  a  historical  review.  Most  of  the  writers  on  general  bacteriology,  who 
make  reference  to  the  history  of  the  subject,  almost  invariably  mention 
the  older  ideas  regarding  spontaneous  generation  as  being  the  forerunners 
of  the  modern  ideas  of  bacteriology.  It  is,  however,  the  ancient  theories 
and  beliefs  pertaining  to  the  cause  of  decay,  disease,  and  epidemics  which 
are  even  more  directly  associated  with  the  first  more  important  discoveries 
pertaining  to  modern  bacteriological  pathology. 

For  the  purposes  of  simplification,  condensation,  and  greater  clearness 
the  historical  review  is  divided  into  periods  or  epochs.  It  is  not  possible, 
in  the  following  brief  outline,  to  cite  all  investigations  of  importance. 
Only  a  few  of  the  epoch-making  specialists  are  mentioned. 

Period  I 

From  Hippocrates  (300  B.  C.)  to  Leeuwenhoek  (1656).  (The  earliest 
ideas  regarding  epidemics  and  spontaneous  generation.) 

From  the  earliest  times  the  more  scholarly  writers  mentioned  certain 
noxious  gaseous,  and  odoriferous  substances  or  efHuvias  as  being  the  cause 
of  epidemics.  These  effluvias  were  supposed  to  emanate  from  the  soil, 

5 


6  PHARMACEUTICAL   BACTERIOLOGY 

from  the  air,  from  water,  stagnant  pools,  marshes,  from  decaying  and 
putrescent  substances,  from  crowded  habitations,  army  cami  'tc.  The 
common  people  throughout  the  world  and  throughout  all  age:  have  held 
the  belief  that  pestilence  and  disease  was  the  manifestation  of  divine  or 
supernatural  influence,  the  judgment  of  an  angry  deity,  a  punishment 
inflicted  on  mankind  for  their  sins  and  iniquities,  beliefs  which  are  oc- 
casionally asserted  even  at  the  present  time.  Changes  of  season,  climatic 
conditions,  and  the  influence  of  heavenly  bodies  were  also  considered  as 
causative  of  diseases  of  an  epidemic  nature. 

Animals,  such  as  rats,  mice,  and  insects,  have  long  been  recognized  as 
possible  carriers  of  disease.  An  English  investigator  has  recently  dis- 
covered some  very  excellent  sanitary  rules  in  the  Vedas  of  the  Hindus. 
The  following  is  a  translation  from  Book  VI,  verse  50,  of  the  Atharva- 
Veda. 

"Destroy  the  rat,  the  mole,  the  boring  beetle;  cut  off  their  heads,  O  asvins. 

"Bind  fast  their  mouths;  let  them  not  eat  our  barley;  so  guard  ye  twain  our  growing 
corn  from  danger. 

"  Hearken  to  me,  lord  of  the  female  borer,  lord  of  the  female  grub !  Ye  rough-toothed 
vermin. 

"Whate'er  ye  be,  dwelling  in  woods,  and  piercing,  we  crush  and  mangle  all  those 
piercing  insects." 

By  " piercing  insects"  no  doubt  mosquitos  are  meant.  If  the  injunc- 
tions were  literally  obeyed,  plague,  malaria,  and  certain  protozoic  diseases 
would  be  abolished  from  India. 

Hippocrates  (460-377  B.  C.),  the  father  of  medicine,  considered  seasons 
and  winds  as  the  cause  of  pestilence,  particularly  the  long  continued  south- 
erly winds  (for  Greece),  and  a  warm,  humid,  clouded  atmosphere.  Galen 
(130-220  A.  D.)  held  similar  beliefs.  He  declared  that  diseases  arose  from 
a  putridity  of  the  air  or  from  atmospheric  and  weather  conditions.  Mar- 
cellinus  (359  A.  D.),  a  warrior  as  well  as  philosopher  and  historian,  declared 
that  the  decomposing  bodies  left  on  the  battlefield  were  the  cause  of  "  pesti- 
lential distempers,"  also  caused  by  extremes  in  weather,  by  marsh  effluvias, 
violent  heat,  and  a  vitiated  atmosphere.  Aetius  (fifth  century),  an  emi- 
nent physician,  declared  that  epidemics  or  common  diseases  were  caused 
by  bad  food,  bad  water,  immoderate  grief,  hunger,  excesses,  particularly 
abundance  following  extreme  want,  lack  of  exercise,  excessive  humidity, 
and  putrid  substances.  Alpinus,  a  Venetian  physician  of  the  sixteenth 
century,  explained  how  the  cause  of  plagues  and  epidemics  may  be  carried 
by  persons  or  in  cargoes.  He  pointed  out  that  a  given  disease  from  one 
country  is  more  malignant  than  the  same  disease  from  another  country. 
During  the  dark  and  middle  ages  various  ecclesiastical  and  lay  writers 
ascribed  epidemics  and  pestilence  to  a  variety  of  causes — the  wrath  of 


HISTORICAL  7 

God,  to  demons  or  evil  spirits,  comets,  meteors,  earthquakes,  volcanic 
eruptions,  cyclones,  eclipses  of  the  sun,  terrific  storms,  wars,  famines, 
great  fires,  etc.  Even  as  late  as  1799  no  less  an  authority  than  Noah 
Webster  makes  the  following  declaration:  "All  the  great  plagues  which 
have  afflicted  mankind  have  been  accompanied  with  violent  agitations  of 
the  elements.  The  phenomenon  most  generally  and  closely  connected 
with  pestilence  is  an  earthquake.  From  all  the  facts  which  I  can  find  in 
history,  I  question  whether  an  instance  of  any  considerable  plague,  in 
any  country,  can  be  mentioned  which  has  not  been  immediately  preceded 
by,  or  accompanied  with,  convulsions  of  the  earth.  If  any  exceptions 
have  occurred,  they  have  escaped  my  researches.  It  does  not  happen 
that  every  place  where  pestilence  prevails  is  shaken;  but  during  the  prog- 
ress of  the  disease  which  I  denominate  pestilence,  and  which  runs,  in  cer- 
tain periods,  over  large  portions  of  the  globe,  some  parts  of  the  earth, 
and  especially  those  which  abound  most  with  subterranean  fire,  are 
violently  agitated."  Were  Noah  Webster  alive,  he  would  certainly  cite 
the  recent  plague  on  the  Pacific  Coast  as  bearing  out  his  assertions.  On 
April  1 8,  1906,  the  coast  region  about  San  Francisco  was  certainly "  vio- 
lently agitated,"  and  this  phenomenon  was  followed  by  the  plague  (black 
pest,  bubonic  plague).  But  what  were  the  actual  facts?  The  plague 
had,  in  all  probability,  ^existed  in  a  sporadic  form  in  "  Chinatown,"  in 
San  Francisco,  and  in  other  places  on  the  Pacific  coast  for  many  years. 
In  1903  several  authentic  cases  came  to  notice  and  were  reported.  The 
reasons. why  the  disease  had  not  previously  gained  a  stronger  foothold  in 
San  Francisco  are  several.  Chinatown  is  more  or  less  isolated  (socially, 
at  least)  from  the  rest  of  the  city,  and  the  poorer,  more  filthy  class  of  the 
Chinese  do  not  as  a  rule  mingle  with  the  white  population.  The  disease 
is  an  Oriental  filth  disease.  After  the  earthquake  and  fire  of  April  18-22, 

1906,  the  Chinese  of  all  classes,  the  plague-infected  rats  and  fleas  of  the 
Chinese  quarters,  became  thoroughly  intermingled  with  the  rest  of  the 
stricken  population,  and  as  a  result  there  were  established  several  new  foci 
of  plague  infection,  which  accounted  for  the  increase  in  plague  cases  in 

1907,  a  condition  which  was  soon  under  control,  thanks  to  the  strenuous 
efforts  of  the  federal  government,  the  board  of  health,  and  various  citizens, 
organizations. 

Several  writers  of  remote  times,  as  well  as  occasional  writers  of  the  dark 
and  middle  ages,  held  the  opinion  that  the  cause  of  disease,  the  disease- 
producing  efiiuvias,  might  be  carried  long  distances  by  air  currents,  in 
ships,  or  by  caravans,  and  that  the  poison  may  enter  the  system  via  the 
air  passages,  through  the  skin,  or  through  the  digestive  tract.  Hodges, 
an  Englishman,  who  wrote  a  treatise  on  the  London  plague  of  1665,  de- 
clared that  some  essential  alteration  in  the  air  is  necessary  to  the  propaga- 


8 


PHARMACEUTICAL  BACTERIOLOGY 


tion  of  this  disease.  That  is,  the  "nitro-aerial"  principle,  which  causes 
or  invigorates  plant  and  animal  life,  is  supposed  to  become  vitiated. 

The  corrupting  principle  is  a  " subtle  aura  or  vapor"  which  is  "extri- 
cated from  the  bowels  of  the  earth."  This  plague-causing  poison  was  said 
to  affect  trees  and  other  plants,  fishes  and  other  animals,  as  well  as  man. 
Dr.  Mead  declared  that  epidemics  were  caused  by  (i)  diseased  persons,  (2) 
goods  imported  from  infected  places,  and  (3)  a  vitiated  or  poisoned  state  of 
the  air,  notions  which  may  be  considered  as  the  direct  forerunners  of  the 
germ  theory  of  disease. 

Let  us  now  go  back  and  consider  the  ancient  ideas  regarding  spontane- 
ous generation.  Anaximander,  of  Miletus,  who  lived  during  the  forty- 
third  Olympiad  (610  B.  C.),  believed  that  many  animals  developed de now, 
from  moisture  and  water  acted  upon  by  sun  and  warmth.  The  extremist. 


FIG.  i. — Fronrthe  Arcana  Natures  of  A.  van  Leeuwenhoek.  The  first  published 
illustration  of  bacteria.  These  bacilli  of  the  mouth  cavity  were  seen  with  the  aid  of  sim- 
ple lenses  only,  a,  b,  bacilli;  c,  a  spirillum;  e,  perhaps  chain  forms  of  bacilli;  d,  illustrat- 
ing the  characteristic  motion  of  certain  bacilli  (n  to  m). 

Empedocles  of  Agrigentum  (450  B.  C.),  declared  that  all  living  things  upon 
the^earth  were  capable  of  originating  spontaneously.  Aristotle  (384  B.  C.) 
taught  that  some  plants  and  animals  originated  spontaneously.  Ovid, 
some  three  centuries  later,  gives  instructions  how  to  create  bees  spon- 
taneously in  the  carcasses  of  horses.  To  within  recent  times  the  belief 
that  certain  animals  could  originate  spontaneously,  that  is,  without  a  pre- 
existing parent,  was  quite  general,  and  differed  only  in  grotesqueness. 
Cardan  as  late  as  1542  declared  that  water  created  fishes,  and  that  many 
fermentative  processes  created  animals.  Van  Helmont  gives  instructions 
how  to  produce  mice  artificially.  Kircher  boldly  declared  that  he  had 
seen  certain  animals  develop  spontaneously  before  his  eyes.  Paracelsus 
gives  instructions  how  to  make  homunculi.  The  instructions  are  quite 


HISTORICAL  9 

simple.  Certain  substances  are  placed  in  a  bottle,  the  bottle  is  well 
stoppered  and  buried  in  a  manure  heap.  Every  day  certain  incantations 
must  be  pronounced  over  the  bottle  in  the  manure  heap.  In  time,  Para- 
celsus declared,  a  small  living  human  being  (homunculus)  will  appear  in 
the  bottle.  Paracelsus,  however,  naively  admits  that  he  has  never  suc- 
ceeded in  inducing  the  homunculus  to  continue  alive  after  being  taken  from 
the  bottle.  Gradually  these  grotesque  and  extreme  opinions  regarding 
tpontaneous  generation  were  abandoned,  and  it  was  declared  that  only 
she  lower  plants  and  animals,  such  as  seaweeds,  algae,  lichens,  lice,  mites, 
maggots,  etc.,  could  develop  spontaneously.  In  fact,  we  can  find  fairly 
intelligent  individuals  to-day  who  firmly  believe  that  certain  animals,  as 
lice,  mites,  etc.,  can  originate  without  a  parent,  and  that  the  hair  from  the 
tail  or  mane  of  a  horse  will  change  into  a  worm  or  snake  if  placed  in  a  bottle 
of  water  and  exposed  to  light  and  warmth. 

From  the  earliest  records  we  learn  that  the  value  of  disinfectants  in  pre- 
venting the  spread  of  infectious  diseases  (epidemics  and  plagues)  was 
known.  Ovid  states  that  the  shepherds  of  his  time  used  burning  sulphur 
for  bleaching  wool  and  to  free  it  from  infectious  diseases.  In  time  of 
plagues,  big  fires  were  made  to  stay  the  ravages  of  pestilential  diseases. 
The  Mosaic  law  is  replete  with  instructions  regarding  cleanliness  as  a 
means  of  preventing  disease.  Wine  was  highly  valued  as  a  dressing  for 
wounds,  having  the  effect  of  preventing  or  checking  pus  formation. 

Period  II 

From  Leeuwenhoek  (1656)  to  Schwann  (1837).  (Discovery  of 
micro-organisms  and  the  early  investigations  regarding  their  activities.) 

As  early  as  1646  Kircher  suggested  that  certain  diseases  might  be  due 
to  very  minute  organisms  which  were  supposed  to  originate  spontaneously 
under  certain  conditions.  Anton  van  Leeuwenhoek  is  very  justly  called 
the  father  of  microscopy,  and  to  him  must  undeniably  be  given  the  credit 
of  first  having  discovered  and  actually  figured  microbes  and  other  micro- 
organisms. His  Arcana  Natura  was  published  in  1656  in  four  volumes. 
It  is  a  most  interesting  work,  and  con  tains  many  good  illustrations  showing 
microbes  of  the  mouth  cavity,  infusoria  of  stagnant  water  and  cellular 
structure  of  vegetable  tissues.  He  observed  the  motion  of  bacteria  and  in- 
fusoria, made  measurements,  illustrated  capillary  circulation  in  the  web 
of  the  frog's  foot,  etc.  He  was  closely  followed  by  Robert  Hooke,  who 
published  his  Micrographia  in  1658.  The  discoveries  of  Leeuwenhoek 
and  Hooke  were  certainly  epoch-making.  A  new  world  of  minute  organ- 
isms was  made  known,  the  question  of  spontaneous  generation  received  a 
new  turn,  and  the  way  to  the  discovery  of  the  causes  of  disease  and  fer- 


10  PHARMACEUTICAL  BACTERIOLOGY 

mentation  was  paved.  In  1660  Leeuwenhoek  discovered  yeast  cells. 
From  1660  to  1760  the  microscope  was  actively  employed  by  a  few  investi- 
gators, and  additions  were  slowly  made  to  the  list  of  micro-organisms. 
Audry  (1701)  designated  microbes  worms.  Mliller  of  Copenhagen  (1786), 
grouped  them  under  two  divisions,  monas  and  vibrio.  In  1743  Henry 
Baker,  of  England,  published  his  work,  "The  Microscope  Made  Easy," 
from  which  it  would  appear  that  very  little  progress  had  been  made  since 
the  time  of  Leeuwenhoek  (1656). 

As  early  as  1686  Franceso  Redi  doubted  that  maggots  were  generated 
de  novo  in  putrid  meats.  He  noticed  that  the  presence  of  the  maggots  was 
preceded  by  swarms  of  flies  which,  he  concluded,  had  something  to  do  with 
the  development  of  the  maggots.  He  found  that  meat  from  which  the  flies 


PIG.  2. — From.  Arcana  Natures.     Cell  structure  of  cork.     Cell-lumen  is  shaded  and  cell- 
walls  are  shown  light. 

were  excluded  by  means  of  paper  or  a  very  fine  mesh  wire  screen,  simply 
decayed  without  any  development  of  maggots.  The  paper  cover  and  the 
fine  screen  kept  the  eggs  of  the  flies  from  being  deposited  on  the  meat,  and 
the  meat  was  not  infested  by  maggots,  which,  as  Redi  rightly  conjectured, 
developed  from  the  eggs  of  the  fly-like  imago.  This  very  simple  but  reli- 
able experiment  did  much  to  create  doubt  as  regards  the  correctness  of  the 
theory  of  spontaneous  generation  and  other  related  beliefs. 

Spallanzani  (1777)  was  among  the  first  to  demonstrate  experimentally 
that  boiling  and  hermetically  enclosing  fermentable  liquids  prevented  fer- 
mentation. Ehrenberg  (1828)  discovered  microscopic  organisms  in  dust 
and  in  water,  and  in  1833  he  classified  all  known  bacteria  under  four  orders, 
bacterium,  vibrio,  spirillum,  and  spirocheta.  Cagniard-Latour  and 
Schwann  (1836)  discovered  the  vegetable  nature  of  yeast,  and  in  1837 
Schwann  declared  that  yeast  was  the  direct  cause  of  fermentative  changes 
resulting  in  the  liberation  of  alcohol  and  CO2,  and  that  the  causes  of  decay 
were  to  be  found  in  the  atmosphere.  Berzelius  (1827)  declared  that  the 
yeast  cells  were  the  direct  cause  of  fermentation.  F.  Schulze  (1836)  pre- 
vented decay  in  liquids  containing  certain  organic  substances  by  first  heat- 


HISTORICAL 


II 


ing  or  boiling  them  and  excluding  the  air  by  means  of  a  layer  of  oil  or  by 
closing  the  container  with  cotton  and  supplying  it  with  air  which  had  been 
sterilized  by  passing  through  sulphuric  acid.  Braconnot  (1831)  advanced 
the  theory  that  yeast  cells  had  the  power  of  holding,  and  condensing  within 
the  cell-substance,  the  oxygen  of  the  air  and  conducting  it  to  the  substances 
undergoing  fermentation,  resulting  in  the  splitting  up  of  sugar  into  alcohoi 
and  carbonic  acid  gas. 

The  question  of  spontaneous  generation  was  again  discussed  with  re- 
newed energy.  The  belief  that  larger  animals  could  originate  de  novo  was 
quite  generally  abandoned,  but  it  was  very  persistently  argued  that  micro- 
organisms, maggots  and  a  few  other  very  small  animals  could  thus  develop. 
Bastian  was  perhaps  the  leader  in  the  arguments  in  favor  of  spontaneous 
generation,  opposed  by  Schwann,  Pasteur,  and  others.  Schroeder  and  von 
Dusch  demonstrated  that  decay  could  be  prevented  by  boiling  and  sup- 


FIG.  3. — Flask,  containing  an  organic  substance,  a,  hermetically  closed  by  means  of 
a  stopper,  b.  The  bent  tube  is  open  at  e,  admitting  air.  Dust  and  microbes  lodge  at 
the  bends  d  and  c. 

plying  air  that  had  been  filtered  through  cotton.  Pasteur  (1862)  used 
bent  tubes  to  supply  air  to  the  previously  sterilized  (by  heating)  substance, 
as  shown  in  Fig.  3.  The  microbes  in  the  air  passing  through  the  tube  are 
deposited  (by  gravity)  in  the  lower  bends  of  the  tube.  Those  favoring  the 
theory  of  spontaneous  generation  nevertheless  continued  their  arguments. 
It  was  pointed  out  that  changes  of  decay  took  place  in  eggs,  in  internal 
tissues  and  organs  of  the  dead  as  well  as  in  the  living,  etc.,  where,  it  was 
supposed,  microbes  could  not  possibly  have  access.  However,  further 
convincing  experiments  gradually  silenced  all  opposition.  Bastian  and 
a  few  followers  took  practically  their  last  stand  in  1875,  and  since  that  time 
no  scientist  of  repute  has  ever  argued  in  favor  of  spontaneous  generation, 
though  the  question  of  the  primal  origin  of  living  things  remains  un- 
answered. 


12  PHARMACEUTICAL  BACTERIOLOGY 

Vaccination  as  a  protection  against  virulent  small-pox  was  practised 
early  in  the  eighteenth  century  in  Turkey  and  other  Oriental  countries,  and 
was  introduced  into  Europe  ma  England  through  the  influence  of  Lady 
Mary  Wortley  Montagu.  A.  von  Humboldt  stated  that  the  Mexicans 
practised  vaccination  at  a  very  early  period.  This  early  vaccination  mate- 
rial was  obtained  from  a  pustule  of  a  small-pox  patient,  and  not  from  the 
cow,  as  at  present.  The  immunity  against  subsequent  attacks  was  es- 
tablished, but  the  disease  transmitted  through  this  older  method  of  vacci- 
nation was  severe  and  often  fatal;  besides,  the  general  vaccination  was  a 
source  of  spreading  the  disease.  In  1840  this  form  of  vaccination  was 
prohibited  in  England  by  act  of  Parliament. 

-.  In  1768  Jenner's  attention  was  attracted  to  the  value  of  vaccination, 
and  after  a  series  of  patient  researches  he  perfected  the  method  of  vaccina- 
tion by  means  of  the  virus  obtained  from  a  cow  which  had  been  inoculated 
with  small-pox  (vaccinia).  Jenner  established  the  first  public  institution 
for  vaccination  in  1799,  and  in  the  following  year  the  practice  was  intro- 
duced into  France,  Germany,  and  the  United  States.  Vaccination  with 
•vaccinia  material  is  now  universal  in  all  civilized  countries  and  in  countries 
under  civilized  control,  and  as  a  result  small-pox  in  an  epidemic  form  does 
not  occur  in  these  countries,  and  the  disease  has  become  less  and  less  viru- 
lent, so  that  it  is  no  longer  the  dreaded  scourge  that  it  was  two  centuries 
ago.  In  spite  of  the  beneficient  influence  of  vaccination,  there  are  indi- 
viduals who  oppose  this  simple,  harmless  operation  with  all  the  energy  that 
ignorance  is  capable  of.  Civilized  countries  are  beginning  to  raise  the 
long-enforced  small-pox  quarantine  as  a  wholly  unnecessary  infliction, 
because  vaccination  makes  the  spreading  of  small-pox  impossible.  France 
has  raised  the  quarantine,  and  so  have  several  other  countries,  examples 
which  will  no  doubt  soon  be  followed  generally.  In  conclusion,  it  is  of 
interest  to  note  that  the  primary  cause  of  small-pox  is  unknown  even  to 
this  day.  Ho  organism  has  thus  far  been  isolated  from  diseased  tissues 
to  which  small-pox  manifestations  could  be  ascribed. 

Period  III 

From  Schwann  (183?)  to  Pasteur  (1862).  (Investigations  per- 
taining to  the  relationship  of  micro-organisms  to  fermentation  and  disease. ) 

The  discoveries  of  the  cause  of  fermentation,  of  decay,  and  of  wound 
infection,  are  closely  associated.  Many  centuries  ago  Varro  expressed  it  as 
his  opinion  that  certain  minute  animals,  breeding  in  marshy  places,  got 
into  the  system  through  mouth  and  nostrils  and  caused  the  disease  and 
decay  of  the  tissues.  Theodoric  (1260)  taught  that  wound  infection  came 
from  the  air.  To  prevent  such  infection  he  applied  wine,  which  is  known 


HISTORICAL  13 

to  be  somewhat  antiseptic.  John  Colbach  (1704)  described  a  "new  and 
secret  method  of  treating  wounds  by  which  healing  took  place  without 
inflammation  or  suppuration." 

From  earliest  time  up  to  as  late  as  1860,  it  was  quite  generally  taught 
that  all  normal  healing  of  wounds  and  cuts  must  be  preceded  by  pus-for- 
mation. A  "laudable  pus"  was  recognized,  the  presence  of  which  was 
looked  upon  as  a  hopeful  sign  and  indicated  that  repair  was  proceeding 
favorably.  If  the  laudable  pus  which  was  of  a  whitish  creamy  consist- 
ency changed  to  a  watery  consistency,  it  was  considered  an  unfavorable 
sign. 

After  Schwann  and  others  had  demonstrated  that  fermentation  was  due 
to  the  presence  of  yeast  cells,  and  it  was  proven  conclusively  that  decay 
was  caused  by  bacteria,  the  relationship  of  bacteria  to  disease  began  to  re- 
ceive consideration.  Rayner  and  Devaine  (1850)  found  bacterial  rods  in 
animals  suffering  from  splenic  fever.  As  early  as  1840  Henle,  who  is  by 
some  considered  the  father  of  modern  bacteriology,  made  some  very 
valuable  deductions  regarding  the  relationship  of  micro-organisms  to  dis- 
ease. He  recognized  a  "contagium"  (the  active  cause  of  the  disease 
associated  with  micro-organisms),  which  was  supposed  to  be  air-like  arid  yet 
at  the  same  time  fixed.  It  was  supposed  to  retain  its  activity  for  years  in 
the  dry  state.  An  unweighable  and  unmeasurable  quantity  of  this  sub- 
stance may  cause  an  extensive  epidemic.  Air  currents  can  carry  the  con- 
tagium great  distances  and  cause  epidemics  in  widely  separated  areas. 
Bassi  (1835)  declared  that  a  fungus  was  the  cause  of  the  muscardine  dis- 
ease of  silkworms.  Pollender  (1855)  reported  that  bacteria  caused  anthrax, 
verified  by  Devaine  in  1863.  Hallier,  an  enthusiast  but  not  reliable  as  an 
investigator,  declared  that  scarlet  fever,  measles,  typhus,  and  cholera  were 
caused  by  bacteria.  His  deductions  were,  however,  not  based  upon  scien- 
tific research  and  proof.  Rindfleisch  (1866)  and  Waldeyer  (1868)  gave 
considerable  attention  to  wound  infection,  which,  they  declared,  was  due 
to  microbic  invasion.  In  1869  Pasteur  demonstrated  the  microbic  cause 
of  the  silkworm  disease  which  interfered  very  seriously  with  the  silk 
industry  in  France.  Pasteur  and  Klebs  demonstrated  experimentally 
that  bacteria  could  be  grown  in  artificial  culture  media,  and  Robert 
Koch  proved  that  the  pathogenic  microbes  actually  secreted  the  disease- 
causing  substance.  This  was  demonstrated  by  transferring  an  infinitely 
small  quantity  of  the  germ  material  from  a  diseased  organ  to  a  suitable 
culture  medium  and  making  sub-cultures,  until  the  last  culture  must  con- 
tain less  than  the  trillionth  part  of  the  original  substance.  Nevertheless, 
inoculations  from  the  last  culture  developed  the  disease  withfull  energy. 
This  experiment  was  made  to  meet  the  assertions  that  the  cause  of  the  dis- 
ease did  not  reside  in  the  bacterium,  and  that  [the  bacterium,  if  present 


14  PHARMACEUTICAL  BACTERIOLOGY 

in    the   disease,    was   merely  incidental    to  and  not   causative   of  the 
disorder. 

A  heated  controversy  continued  for  some  time.  Such  authorities  as 
Liebig,  Nageli,  Bastian,  Cohn,  Billroth,  Hiller,  Schroeder,  Hoppe-Seyler, 
Ktihne,  Tiegel,  Sanderson,  Nencki,  Serval,  and  Paschutin  declared  that 
micro-organisms  were  not  the  cause  of  decay,  fermentation,  and  disease; 
that  these  changes  were  due  to  chemical  substances.  However,  such  men 
as  Pasteur,  Koch,  Panum,  Klebs,  and  others  forged  link  after  link  in  the 
chain  of  evidence  connecting  the  causative  relationship  of  bacteria  to 
disease. 

Period  IV 

From  Pasteur   (1862)   to  Behring    (1890).     (Period  of  remarkable 
activity  in  pathological  bacteriology.) 

It  would  be  impossible  in  a  brief  review  to  cite  all  of  the  important 
investigations  of  this  period.  Pasteur,  Koch,  and  others  had  already  given 
the  subject  of  bacteriological  technic  considerable  attention.  The  most 
suitable  culture  media,  laboratory  apparatus,  stains,  etc.,  were  determined. 
The  compound  microscope  had  now  reached  a  high  degree  of  perfection, 
and  the  oil-immersion  lenses  made  the  closer  study  of  the  morphology  of 
bacteria  possible. 

As  might  be  expected,  the  importance  of  germicides  in  surgery  received 
first  attention.  The  " laudable  pus- "  formation  ideas  were  abandoned.  It 
became  the  surgeon's  duty  to  induce  "primary  union"  or  healing  by  " first 
intention,"  that  is,  healing  without  any  pus  formation  whatever.  This 
demanded  that  the  surfaces  of  the  incision  be  brought  in  close  contact,  and 
that  all  bacterial  infection  be  prevented  by  the  use  of  antiseptic  dressings, 
antiseptic  solutions  in  the  form  of  irrigations  and  sprayings,  etc.  Sir 
Joseph  Lister,  of  Scotland  (1875),  brought  the  use  of  disinfectants  in  sur- 
gery to  a  high  degree  of  perfection,  and  modern  antiseptic  surgery  is 
often  designated  "Listerism."  The  chief  antiseptic  of  Lister  and  his  fol- 
lowers was  carbolic  acid,  which  was  used  for  free  wound  irrigation  and 
general  disinfection.  He  operated  in  a  spray  of  carbolic  acid  solution.  As 
late  as  1890  there  was  to  be  found  an  occasional  lecturer  in  a  college  of 
medicine  who  held  out  against  the  germ  theory,  and  not  a  small  number  of 
the  eminent  opponents  mentioned  in  the  previous  period  carried  their 
mistaken  notions  with  them  to  the  grave. 

The  name  of  Robert  Koch  will  stand  throughout  the  ages  as  the  leader 
in  modern  bacteriological  science.  Early  in  life  he  was  convinced  of  the 
correctness  of  the  germ  theory  of  disease,  but  his  first  contributions  to 
bacteriological  science  awakened  a  storm  of  opposition .  Billroth,  of  Vienna, 
and  others  persisted  in  declaring  that  microbes  were  not  causative  of  pus- 


HISTORICAL  15 

formation  or  of  the  development  of  disease;  but  that  microbes  might  be 
accidentally  present,  due  to  the  action  of  a  "phlogistic  zymoid "  which 
developed  in  the  animal  organism. 

In  1882  the  French  government  sent  a  medical  commission  to  India  to 
determine  if  possible  the  cause  of  Asiatic  cholera,  but  the  commission  re- 
turned with  a  negative  report  as  far  as  a  bacterial  cause  of  the  disease  was~ 
concerned.  In  1883  the  German  government  sent  a  similar  commission, 
headed  by  Robert  Koch,  and  the  report  of  this  commission  was  that  Asiatic 
cholera  was  caused  by  a  bacillus,  the  famous  comma  bacillus  of  Koch. 
The  work  of  Koch  in  connection  with  the  study  of  cholera  seemed  to  act 
as  a  wonderful  stimulus  and  other  eminent  investigators  made  important 
discoveries  within  the  year  or  two  following.  Klebs  and  Loffler  discovered 
the  diphtheria  bacillus  in  1884.  Fraenkel,  Weichselbaum  and  Fried- 
lander  discovered  the  pneumococcus  in  1884.  Nicolaier  and  Kitasato  dis- 
covered the  tetanus  bacillus  in  1884.  Loffler  and  Schiitz  discovered  the 
glanders  bacillus  in  1882,  and  the  bacillus  of  hog  erysipelas  (Rothlauf)  in 
1885. 

Pasteur*  in  1881  made  his  first  experiments  in  reproducing  rabies  in 
susceptible  animals  by  inoculation  with  material  obtained  from  the  spinal 
cord,  medulla  oblongata,  and  lobes  of  the  brain  of  animals  dead  from 
rabies.  In  1884  he  reported  his  experiments  pertaining  to  the  modification 
of  the  virulence  of  rabies  by  successive  inoculations  into  susceptible  ani- 
mals. His  use  of  this  modified  rabies  virus  as  a  means  of  preventing  a 
severe  and  fatal  course  of  the  disease  in  those  bitten  by  animals  suffering 
from  hydrophobia,  is  familiar  to  all.  Thousands  of  cases  have  been  treated 
successfully  at  Pasteur  institutes  established  throughout  the  larger  cities 
of  the  civilized  world. 

The  above  are  only  a  few  of  the  important  investigations  of  this  period. 
The  causative  relationship  of  microbes  to  certain  diseases  was  undeniably 
established.  The  voices  of  opposition  were  silenced. 

This  period  is  especially  notable  for  the  development  of  antiseptic 
surgery.  As  a  result,  operations  were.no  longer  dreaded  as  in  former  times. 
Fatal  infections  following  operations  now  became  rare.  Thousands  of 
lives  are  saved.  To  remove  or  destroy  the  pus  germs  in  open  wounds  or  to 
prevent  the  access  of  germs  to  wounds,  cuts,  and  abrasions,  has  become  a 
simple  matter,  a  simple  mechanical  application  of  suitable  antiseptics. 

The  progress  of  purely  medical  bacteriology  was  not  so  marked.  Al- 
though it  was  proven  that  certain  diseases  were  due  to  bacteria,  there  were 
no  satisfactory  means  of  destroying  them  in  the  system.  Internal  anti- 
septics were  tried,  but  without  satisfactory  results,  as  a  rule.  However, 
preventive  medicine  based  on  a  bacteriological  knowledge  gave  good 
results. 


1 6  PHARMACEUTICAL  BACTERIOLOGY 

Period  V 

From  Behring  (1890)  to  Wright  (1907).  (Discovery  of  serum  therapy, 
bacterial  vaccines,  and  development  of  utilitarian  bacteriology.) 

The  subject  of  immunity  from  disease  received  early  attention.  Age 
immunity,  race  immunity,  animal  immunity,  individual  immunity,  artifi- 
cial immunity,  natural  immunity,  acquired  immunity,  etc.,  attracted 
attention  and  received  careful  consideration.  Metchnikoff  (1884)  ex- 
plained immunity  on  the  supposition  that  certain  white  corpuscles  (leuco- 
cytes, phagocytes)  of  the  blood  devoured  the  microbes  which  entered  the 
system.  These  white  blood  corpuscles  are  the  guardians  of  health.  They 
attack  and  feed  upon  any  disease  germs  which  may  enter  the  body,  either 
via  the  digestive  tract,  the  respiratory  tract,  or  via  the  circulatory  system. 
If  the  leucocytes  are  deficient  in  number,  or  if  the  microbes  are  excessive 
in  number,  disease  will  develop.  This  theory  had  numerous  followers,  as 
well  as  opponents.  It  is  now  generally  accepted  as  correct,  borne  out  by 
observation  and  by  experimental  evidence. 

The  next  important  discovery  was  that  blood  serum  had  bactericidal 
properties  in  a  varying  degree,  and  that  in  addition  to  this  there  was  some- 
thing in  the  blood  which  had  a  tendency  to  neutralize  or  destroy  the  action 
of  the  poisons  or  toxins  formed  by  pathogenic  microbes.  No  one  par- 
ticular bacteriologist  can  be  said  to  have  made  these  discoveries.  We  can 
only  name  a  few  of  the  leading  investigators  who  worked  along  these  lines, 
leading  to  the  discovery  of  the  relationship  of  immunity  and  antitoxins — 
Behring,  Brieger,  Buchner,  Calmette,  Chamberland,  Ehrlich,  Emmerich, 
Fliigge,  Frankel,  Hiieppe,  Jetter,  Kitasato,  Klemperer,  Loffler,  Rankin, 
Roux,  Wassermann,  and  others.  These  eminent  authorities  have  demon- 
strated the  possibility  of  developing  or  aiding  the  antitoxic  or  immunizing 
power  of  the  blood  or  of  the  body  cells  by  introducing  sera  obtained  from 
the  blood  of  animals  in  which  the  antitoxic  power  is  naturally  high  or  is 
made  so  as  the  result  of  special  treatment.  Numerous  sera  (containing 
antitoxins  and  toxins)  were  tried ;  the  one  which  first  proved  entirely  satis- 
factory was  the  diphtheria  antitoxin  of  Behring,  which  is  now  in  universal 
use.  Others  are  used  more  or  less  successfully,  and  some  are  still  in  the 
experimental  stage. 

In  1890  Koch  reported  on  a  "lymph"  to  be  used  in  the  treatment  of 
tuberculosis.  This  lymph  was  a  glycerin  extract  of  the  toxin  of  the  bacillus 
of  tuberculosis,  and  was  to  be  used  in  the  treatment  of  this  dread  disease, 
but  the  hopes  of  Koch  were  not  realized,  as  the  remedy  proved  a  failure, 
and  it  soon  fell  into  disuse,  to  be  again  taken  up  very  recently.  In  1907 
Wright  made  knownhi  s  discovery  of  opsonins.  According  to  this  authority, 
there  exist  in  the  blood  certain  substances  which  have  the  power  of  acting 


HISTORICAL  1 7 

on  the  invading  bacteria  in  such  a  manner  as  to  render  them  more  liable  to 
be  attacked  and  assimilated  by  the  white  blood-corpuscles  or  leucocytes. 
There  are  possibly  as  many  opsonins  as  there  are  microbes  capable  of  being 
digested  by  the  leucocytes.  The  microbe-devouring  power  of  the  leuco- 
cytes can  be  increased  by  the  use  of  bacterial  vaccines,  which  consist  of 
suspensions  of  microbes.  Very  minute  quantities  are  injected  into-the 
system,  and  the  resulting  reaction  increases  the  power  referred  to. 

Toxins  of  bacterial  origin  received  the  attention  of  investigators,  and 
antibodies  (antitoxins)  were  extensively  discussed  as  to  their  possible  rela- 
tionship to  health  and  disease.  Enzymes,  in  their  relationship  to  life 
processes  in  plants  and  in  animals,  were  investigated.  It  is  now  supposed 
that  soil  toxins  of  plan  t  origin,  as  well  as  those  of  bacterial  origin,  influence 
plant  growth.  Glandular  preparations  (ductless  glands)  have  been  care- 
fully tested,  and  several  of  these  are  in  use. 

As  the  result  of  Wright's  discovery  of  the  use  of  bacterial  vaccines  in 
increasing  the  opsonic  index,  the  tuberculin  (lymph)  of  Koch  was  again 
tried  in  the  treatment  of  tuberculosis,  apparently  with  some  success. 

It  was  found  that  there  were  many  bacteria  other  than  those  which 
caused  disease  in  animals  and  plants.  Some  were  found  to  be  decidedly 
beneficial.  Bacterial  cultures  were  employed  in  butter-making  (ripening 
of  cream),  in  cheese-making,  in  tanning,  in  paper-making,  siloing,  etc. 
Some  bacteria  are  employed  to  exterminate  certain  pest  animals.  A  mi- 
crobic  chintz  bug  exterminator  was  tried  in  189-597,  but  it  proved  a  failure. 
Microbic  rat  and  mice  exterminators  (azoa,  ratite,  mouratus,  etc.)  are  now 
being  tested,  and  they  appear  to  be  quite  succesful,  at  least  in  certain 
localities  and  under  certain  conditions.  A  microbic  rabbit  exterminator 
has  been  tried  in  Australia. 

In  1879  Dr.  Frank,  of  Berlin,  began  his  investigations  of  the  leguminous 
root  nodule  microbes.  In  1893  the  writer  attempted  to  utilize  these  mi- 
crobes in  increasing  the  yield  of  certain  gramineous  crops.  In  1896  Nobbe 
and  Hiltner,  of  Germany,  introduced  a  patented  microbic  fertilizer  for 
leguminous  plants.  In  1907  a  California  soil  microbe  was  isolated  which 
appears  to  be  especially  active  in  promoting  the  growth  of  sugar  beets. 
This  experiment  led  to  the  supposition  that  perhaps  every  species  of  plant 
has  its  peculiar  bacterial  flora,  symbiotically  (mutually  beneficent) 
associated  with  the  root  system,  mutually  essential  to  active  development. 
The  importance  of  soil  bacteria  in  setting  free  plant  foods  has  been  dem- 
onstrated by  numerous  investigators  of  Europe  and  of  the  United  States. 
Yeast  and  mould  organisms  are  practically  utilized  in  the  manufacture  of 
beer,  sake,  and  other  food  and  drink  products. 

The  above  condensed  outline  of  the  history  of  bacteriology  may  be 
summed  up  as  follows: 


s 


1 8  PHARMACEUTICAL  BACTERIOLOGY 

1.  Ancient  conceptions  of  disease  and  of  spontaneous  generation,  dat- 
ing back  to  500  years  B.C. 

2.  Discovery  of  micro-organisms  about  1660  by  Leeuwenhoek,  followed 
by  the  work  of  Robert  Hooke  and  a  few  others. 

3.  Discovery  of  bacteria  in  air,  dust,  and  decaying  substances,  and  the 
causal  relationship  of  microbes  to  decay,  and  of  the  yeast  organisms  to 
fermentation. 

4.  Disproving  the  theory  of  spontaneous  generation,  by  Schwann  and 
others,  about  1840. 

5.  Discovery  of  the  bacterial  origin  of  certain  diseases — 1862  to  1880. 

6.  Introduction  of  small-pox  vaccination  into  England  by  Jenner  in 
1796. 

7.  Development  of  antiseptic  surgery  orListerism — 1875. 

8.  Period  of  great  activity  in  pathological  bacteriology — 1880  to  1890. 

9.  Discovery  of  the  causes  of  immunity  to  disease,  antitoxin  of  diph- 
theria and  other  antitoxins,  serum  therapy,  etc. — 1886  to  1894. 

10.  Introduction  of  the  use  of  certain  bacteria  in  commerce  and  agri- 
culture. 

11.  Discovery  of  opsonins  and  the  use  of  bacterial  vaccines.     Reintro- 
duction  of  Koch's  lymph  in  the  treatment  of  tuberculosis. 

Nothing  epochmaking  has  been  discovered  in  the  micro-biological 
sciences  within  the  last  decade.  Nothing  new  has  developed  during  the 
world  war.  The  cause  of  cancer  remains  unknown.  The  so-caUed  ultra- 
microscopic  organisms  remain  unidentified.  The  one  bright  ray  in  sani- 
tary science  is  the  typhoid  fever  control  by  means  of  the  immunizing 
bacterins.  The  primary  cause  of  influenza  may  soon  be  discovered. 
Useful  Works  of  Reference  to  Bacteriology  and  Related  Topics 

The  following  references  are  selected  for  collateral  reading.  A  few 
of  these  works  are  rare,  and  can  be  found  only  in  some  of  the  leading 
libraries.  A  reading  of  these  and  other  related  works  will  serve  as  a 
supplement  to  this  text-book.  It  is  not  intended  to  imply  that  all  of 
the  works  cited  should  be  procured.  Others  besides  those  mentioned 
may  be  consulted  as  opportunity  presents  itself.  Some  of  them  can  be 
obtained  from  public  libraries;  others  may  be  ordered  through  the  local 
book  deader,  and  a  few  may  be  borrowed  from  professional  friends. 

HENRY  BAKER.     The  Microscope  Made  Easy.    London.     1743- 

Like  the  work  of  R.  Hoke,  this  is  of  great  historical  interest,  and  is  quite  rare. 
Much  of  it  is  a  copy  of  the  work  of  Leeuwenhoek. 

B.    M.  BOLTON  (H.  U.  Williams).     A  Manual  of  Bacteriology.     P.  Blakiston's  Son 
&  Co.,  Philadelphia.     1910. 

A  most  excellent  work  for  medical  students,  also  of  value  to  students  of  pharmacy. 
H.  W.  CONN.  Agricultural  Bacteriology. 

This  is  a  most  excellent  little  work  treating  of  bacteria  in  water,  in  the  soil,  in 


HISTORICAL 

farm  products,  in  the  dairying  industry,  and  in  plants  and  domestic  animals.     It  is 

well  written  in  a  simple,  clear  style. 

H.  W.  CONN.     Bacteria,  Yeasts  and  Moulds  in  the  Home.     Ginn  &  Co.     1903. 

This  is  of  special  value  to  the  pharmacist,  as  the  organisms  described  may  also  be 
found  in  pharmaceutical  preparations. 
H.  W.  CONN.     The  story  of  Germ  Life.     D.  Appleton  &  Co.,     New  York.     1905. 

Very  useful  and  interesting  general  reading  on  bacteriology. 
S.  M.  COPEMAN.     Vaccination,  Its  Natural  History  and  Pathology.     London.     1899. 

Of  historical  interest,  besides  explaining  the  subject  very  fully. 
E.  M.  CROOKSHANK.     Text-book  of  Bacteriology.     Philadelphia.     1897. 

This  is  much  used  as  a  college  text-book  on  bacteriological  technic.  Not  especially 
adapted  for  general  reading.  Would  serve  as  a  laboratory  guide. 

CHAS.   S.  DOLLEY.     The  Technology  of  Bacteria  Investigation.     S.  E.  Casino  &  Co., 
Boston.     1885. 

Good  reference  work  on  bacteriological  technic.     Somewhat  out  of  date. 
DUNHAM,  and  DAKIN.     A  Handbook  on  Antiseptics.     The  MacMillan  Company.  1917. 

A  small  hand  book  on  war  time  antiseptics,  laying  special  stress  upon  the  dakin 
solution.     The  methods  for  testing  antiseptics  are  entered  into  very  briefly.     Water 
sterilization  and  disinfection  and  the  disinfection  of  carriers  is  explained. 
PAUL  EHRLICH  (Chas.  Bolduin).     Collected  Studies  on  Immunity.     John  Wiley  and 
Sons,  New  York.     1906. 

An  extensive  discussion  of  the  theories  pertaining  to  the  action  of  toxins  and  anti- 
toxins.    Ehrlich's  side-chain  theory  is  quite  fully  treated.     The  subject  is  too  technical 
for  the  average  reader,  and  is  of  great  value  only  to  the  specialist  in  this  branch  of  bac- 
teriology. 
DAVID  ELLIS.     Outlines  of  Bacteriology.    London,  New  York  and  Calcutta.     1909. 

An  excellent  English  work  on  general  bacteriology  especially  valuable  from  the  tech- 
nical and  agricultural  standpoints. 

J.  W.  EYRE.     The  Elements  of  Bacteriological  Technic.     W.  B.  Sa'unders  &  Co.,  Phila- 
delphia.    1902. 

An  excellent  laboratory  guide  for  the  use  of  medical,  dental,  and  technical  students, 
and  which  will  serve  many  purposes  of  the  student  of  pharmacy. 
DANIAL  DE  FOE.     History  of  the  Plague  in  London.    London.     1857. 

Of  historical  interest.     Well  written. 

W.  D.  FROST.     A  Laboratory  Guide  in  Elementary  Bacteriology.     MacMillan  Com- 
pany, New  York.     1903. 

An  excellent  laboratory  guide.     It  contains  no  general  information  regarding  bac- 
teria, and  can  be  used  profitably  only  under  the  guidance  of  a  laboratory  instructor. 
W.  H.  HARROCKS.     An  Introduction  to  the  Bacteriological  Examination  of  Water. 
J.  A.  Churchill,  London.     1901. 

Of  value  to  anyone  interested  in  the  bacterial  contamination  of  water  supplies; 
also  useful  for  general  reading. 
ROBERT  HOOKE.     Micrographia.    London.     1665. 

A  very  rare  and  very  interesting  work  treating  of  the  earliest  discoveries  through  the 
use  of  the  microscope.  Some  of  the  illustrations  are  excellent.  Of  great  historical 
value  and  interest.  Can  be  found  only  in  a  few  of  the  larger  university  and  public 
libraries.  In  English. 

L.  O.  HOWARD.     Mosquitoes :  How  They  Live  and  How  They  Carry  Disease.     McClure, 
Phillips  &  Co.,  New  York.     1901. 

Contains  valuable  information  regarding  these  pests  and  how  they  carry  diseases. 
Of  special  value  in  yellow  fever  and  malarial  districts. 


2O  PHARMACEUTICAL  BACTERIOLOGY 

E.  0.  JORDAN.     A  Text-book  of  General  Bacteriology.     W.  B.  Saunders  &  Co.,  Phila- 

delphia.    1908. 
For  medical  students.     Contains  much  information  of  interest  to  the  pharmacist. 

F.  LAFAR  (Salter).     Technical  Mycology.     London.     1903. 

Rather  technical  for  general  reading.     Treats  of  fermentation  and  fermentation 
products,  use  of  yeast  organisms  and  bacteria  in  the  industries,  etc.     Especially  valu- 
able to  those  interested  in  beer-making,  etc.,  the  dairying  industry,  etc. 
MILLARD  LANGFELD.     Infectious  and  Parasitic  Diseases,  Including  Their  Cause  and 
Manner  of  Transmission.     P.  Blakiston's  Son  &  Co.,  Philadelphia.     1907. 

Contains  much  valuable  information  on  preventive  medicine,  sources  of  infection, 
disinfectants  and  disinfection,  animal  parasites,  etc.     Excellent  collateral  reading  for 
the  pharmacist. 
ANTON  VAN  LEEUWENHOEK.     Arcana  Naturae.     Four  volumes.    London.     1656. 

This  is  by  far  the  most  important  historical  work  on  the  use  of  the  microscope.     In 
Latin.     Some  very  good  illustrations.     Very  rare;  found  in  a  few  libraries  only. 
CHARLES  E.  MARSHALL  (and  collaborators).     Micobiology.     P.  Blaskiston'  Son  and 
Company.     1912. 

A  very  readable  and  fairly  complete  guide  to  the  study  of  micro-organisms,  vege- 
table as  well  as  animal.     This  book  should  be  in  the  hands   of  every  student  of 
microbiology. 
K.  C.  MEZ.     Mikroskopische  Wasser  Analyse.     Brrlin.     1898. 

An  excellent  German  work  treating  of  the  bacteriological  investigation  of  drinking 
water  and  sewage  waters. 
GEO.  NEWMAN.     Bacteria.     G.  Putnam's  Sons,  New  York.     1899. 

Treats  of  bacteria  in  industrial  processes,  bacteria  in  public  health,  in  nature,  in 
soil,  etc.     A  very  valuable  work,  excellent  for  general  reading. 
SAMUAL  GATE.  PRESCOTT     Elements  of  Water  Bacteriology.     John  Wiley  and  Sons. 


T.  M.  PRUDDEN.     The  Story  of  Bacteria.     G.  P.  Putnam's  Sons,  New  York.     1889. 

Very  interesting  reading  on  general  bacteriology  and  on  the  relationship  of  bacteria 
to  health  and  disease. 
M.  J.  ROSENAU.     The'Bacteriological  Impurities  of  Vaccine  Virus.     U.  S.  Public  Health 

and  Marine  Hospital  Service'.     Hygienic  Lab.  Bui.,  No.  12.     1903. 
Of  special  interest  to  pharmacists.     It  should  be  borne  in  mind,  however,  that  since 
the  publication  of  this  report  the  methods  of  vaccine  manufacture  have  been  modified 
somewhat,  and  the  figures  and  results  given  may  no  longer  apply. 
M.  J.  ROSENAU.     An  Investigation  of  a  Pathogenic  Microbe  of  Rats  and  Mice  (B.  typhi- 

murium  Danysz.)     Washington,  D.  C.     1903. 

This  treatise  is  also  of  special  interest  to  pharmacists,  as  the  microbe  referred  to  is 
the  active  ingredient  of  several  patented  rat  and  mouse  exterminators  sold  under  pro- 
prietary names  as  Azoa  (Parke,  Davis  &  Co.),  Rattite,  Mouratus  (Pasteur  Co.),  etc. 
These  exterminators  are  still  under  investigation,  testing,  etc.,  and  the  findings  in  the 
above  report  should  not  be  considered  final  or  conclusive. 
M.  J.  ROSENAU.  Preventive  Medicine  and  Hygiene.  D.  Appleton  'and  Com- 

pany.    1917. 
W.  G.  SAVAGE.     The  Bacteriological  Examination  of  Water  Supplies.     Philadelphia. 

1906. 

A  valuable  treatise.     Contains  a  citation  of  the  more  important  literature  on  the 
subject.     An  excellent  laboratory  guide  for  the  specialist. 

DR.     C.     STICH.      Bacteriologie     und    Sterilization    im    Apothekerbetrieb.     Berlin. 
1904. 


HISTORICAL  21 

In  German  only.     Contains  many  valuable  suggestions  but  too  incomplete  and  too 
much  lacking  in  detail  for  the  student. 
E.  R.  STITT.     Practical  Bacteriology,  Blood  Work  and  Animal  Parasitology.     P.  Blakis- 

ton's  Son  &  Co.,  Philadelphia.     1917. 

Primarily  for  medical  students,  especially  those  interested  in  the  parasitology  of  the 
tropics.     Complete  on  methods.     Full  details  regarding  blood  work  and  use  of  hemacy- 
tometer. 
FRED  W.  TANNER.     Bacteriology  and  Mycology  of  Foods.     John  Wiley  and  Sons. 

1919. 

A  fairly  comolete  summary  of  the  bacteriological  methods  employed  in  food  labora- 
tories, including  the  examination  of  disinfectants.     The  direct  methods  of  microanalysis 
are  explained. 
JOHN  TYNDALL.     Floating  Matter  in  the  Air.    London.     1881. 

A  very  interesting  popular  work  on  the  micro-organisms  of  the  air  and  their  rela- 
tionship to  fermentation  and  putrefaction.     For  general  information. 
NOAH  WEBSTER.     A  Brief  History  of  Epidemics  and  Pestilential  Diseases.     Two 

volumes.     Hartford.     1799. 

Of  great  historical  interest,  though  entirely  antiquated  and  of  no  scientific  value. 
GEORGE  CHANDLER  WHIPPLE.     The  Microscopy  of  Drinking  Water.     John  Wiley  and 

Sons.     1914. 
H.  W.  WILEY.     Food  and  Their  Adulteration.     P.  Blakiston's  Son  and  Company. 

1907. 

The  following  are  a  few  selec'  references  to  naval  and  army  sanitation  and  hygiene 
which  will  serve  as  excellent  collateral  reading  for  the  student  of  pharmacy  (course 
in  sanitation). 
JOSEPH  H.  FORD.     Elements  of  field  Hygiene  and  Sanil  ation.     P.  Blakiston's  Son  and 

Company.     1917. 
VALERY  HAVARD.     Manual  of  Military  Hygiene.     (Third  revised  edition.)     William 

Wood  and  Company.     1917. 

JAMES  CHAMBERS  PRYOR.     Naval  Hygiene.     P.  Blakiston's  Son  and  Company.     1918. 
EDWARD  B.  VEDDER.     Sanitation  for  Medical  Officers.     (War  Manual  No.  i.)     Lea 
and  Febiger.     1917. 


i 


CHAPTER  III 

THE  ORIGIN  OF  BACTERIA  AND  OF  OTHER 
MICRO  -ORGANISMS 

In  consideration  of  the  recent  revival  of  the  interest  in  the  question  of 
the  origin  of  living  matter,  a  work  on  bacteriology  would  be  incomplete 
without  a  brief  mention  of  the  subject,  for  the  origin  of  bacteria  is  one  with 
the  origin  of  life.  Despite  the  interest  mentioned  we  must  from  the  outset 
admit  that  the  answer  to  the  riddle  of  life  has  not  yet  been  solved.  Many 
theories  have  been  advanced,  some  apparently  ridiculous  and  wholly 
without  scientific  warrant;  others  interesting  but  not  plausible;  some 
apparently  scientifically  sound  but  lacking  in  one  or  more  essentials  and 
hence  inconclusive.  The  following  are  a  few  of  the  more  important  hyop- 
theses  and  theories. 

i.  Spontaneous  Generation. — This  has  already  been  referred  to  in 
Chapter  II.  According  to  this  idea,  not  only  microbes,  but  highly  com- 
plex organisms,  as  insects,  fish,  mice,  etc.,  might,  under  suitable  surround- 
ings arise  de  now,  without  a  preexisting  parent.  Paracelsus  gave  specific 
instructions  as  to  the  creation  of  the  "homunculus."  These  old  time 
absurdities  need  not  be  entered  into  further.  Until  quite  recently,  the 
belief  in  the  spontaneous  origin  of  simple  forms  of  life  (bacteria,  protozoa, 
lower  algae  and  fungi)  was  common  among  the  leading  scientists.  How- 
ever, the  rapidly  accumulating  evidence  of  scientific  investigations  soon 
proved  the  entire  erroneousness  of  this  belief  also.  As  soon  as  the  scien- 
tific world  became  convinced,  in  the  face  of  incontrovertible  evidence, 
that  life  does  not  originate  spontaneously,  and  that  it  could  not  be  devel- 
oped artificially  in  the  laboratory,  a  tendency  began  to  manifest  itself  to 
dispose  of  the  entire  matter  as  follows.  Although  life  does  not  originate 
spontaneously  at  the  present  time,  it  must  have  originated  spontaneously  at 
some  time  in  the  distant  past.  Conjectures  were  advanced  as  to  what  the 
remarkable  life  giving  conditions  of  this  remote  past  might  have  been, 
but  none  of  the  suggestions  proved  satisfying.  There  was  nothing  tangi- 
ble or  significant  proposed  or  stated,  and  at  the  present  time  this  idea  is 
no  longer  discussed  among  scientists.  It  receives  a  fleeting  mention  in 
the  class  room  perhaps,  but  it  is  no  longer  considered  worthy  of  a  place  in 
scientific  literature.  The  entire  concept  is  scientifically  dry  and  meatless 
and  shall  be  disposed  of  with  this  brief  mention. 

22 


THE    ORIGIN    OF  BACTERIA   AND    OTHER   MICRO-ORGANISMS          23 

2.  The  Vitalistic   Hypothesis. — This   has  also  been  designated  the 
" vital  spark"  theory  or  hypothesis.     According  to  this  concept  it  was 
assumed  that  some  mystical  energy  or  force,  or  unknown  and  unknowable 
power,  gave  rise  to  life.     It  was  supposed  that  the  difference  between  a  bit 
of  dead  organic  substance  and  a  bit  of  living  organic  substance  was  that 
the  living  substance  had  been  activated  by  some  mysterious  stimulus,  the 
vital  spark.     It  was  even  suggested  that  this  vital  spark  might  be  elec^ 
trical  in  nature,  and  some  misguided  scientists,  in  an  attempt  to  harmonize 
biological  science  with  this  essentially  eclesiastical  hypothesis  of  life, 
suggested  that  life  was  due  to  the  action  of  electrical  discharges  upon 
organic  matter.     No  scientist  today  subscribes  to  the  vitalistic  concept  of 
life. 

3.  The  Panspermistic  Theory. — This  is  also  known  as  the  cosmozoic 
theory  and  is  one  of  the  very  latest  attempts  to  explain  the  origin  of 
life  upon  our  particular  planet,  namely  the  earth.     Despite  the  boldness 
and  daring  of  the  theory,  it  is  well  founded  in  the  physical  sciences  and 
it  is  well  worth  while  to  give  it  a  more  detailed  consideration. 

According  to  the  panspermia  or  cosmozoa  theory  there  is  an  inter- 
planetary distribution  of  germs.  Only  within  comparatively  recent  times 
has  the  idea  become  intelligently  or  rationally  formulated.  Flammarion 
suggested  that  most  of  the  planets  were  inhabited.  De  Montlivault 
(1821)  declared  that  the  first  terrestrial  life  came  from  the  moon.  Richter 
(1865)  impressed  by  the  writings  of  Flammarion,  conceived  the  idea  that 
meteors  might  be  the  interplanetary  carriers  of  seeds  and  perhaps  of 
plants  and  smaller  animals.  Ferdinand  Cohn  (1872)  strongly  supported 
the  idea  of  Richter  which  idea  is  beginning  to  receive  serious  attention 
on  the  part  of  scientists  generally. 

When  a  larger  cosmic  body  collides  with  a  planet,  the  impact  causes 
great  disturbance  and  broken  masses  and  particles  are  driven  into  space 
in  all  directions.  The  heat  generated  by  the  impact  would  no  doubt  kill 
all  organisms  in  the  immediate  vicinity  of  the  point  of  impact,  even  should 
they  survive  the  mechanical  shock.  However,  there  can  be  no  doubt  that 
seeds  and  many  of  the  lower  forms  of  life  could  survive  and  these  might 
be  transported  to  some  neighboring  planet.  It  is,  however,  not  conceiv- 
able how  any  of  the  more  highly  organized  plants  and  animals  could  possibly 
survive  such  a  journey.  Suppose  a  large  mass,  bearing  upon  it  plants  and 
insects,  should  become  detached  from  a  planet  and  finally  approach  the 
earth  entering  the  layer  of  atmosphere,  the  heat  generated  by  the  friction 
would  certainly  destroy  all  life  present. 

The  objections  above  set  forth  as  to  the  meteoric  origin  of  higher  pan- 
germs,  such  as  higher  animals,  plants  and  seeds,  does  not  apply  to  bacteria, 
and  other  similar  small  organisms.  Should  a  meteor  or  other  planetary 


24  PHARMACEUTICAL  BACTERIOLOGY 

fragment  having  upon  its  exterior  deposits  of  organic  matter  with  bacteria 
endospores,  and  filterable  viruses,  enter  the  atmospheric  zone  of  a  planet 
like  that  of  the  earth,  the  resistance  offered  by  the  air  would  at  once  re- 
move the  now  absolutely  dry  pulverulent  germ-bearing  portions  of  the 
meteor,  leaving  them  behind  as  a  very  fine  invisible  dust  which  would  be 
very  much  retarded  in  its  motion  toward  the  earth's  surface;  somewhat 
larger  particles  would  follow  faster  and  become  more  or  less  incandescent 
during  their  flight  through  the  layer  of  atmosphere,  constituting  the 
fiery  trail  of  the  meteor.  It  is  highly  probable  that  the  very  finest  par- 
ticles, as  the  germs  measuring  less  than  0.05^.  in  diameter,  would  never 
reach  the  earth,  being  prevented  from  doing  so  by  the  earth's  electronic 
(electro  magnetic)  repulsion.  Some  spores  and  micro-organisms  would, 
however,  finally  reach  the  earth  due  to'  the  action  of  convection  currents, 
electrical  action  and  the  direct  and  reflected  repulsion  energy  (radiation 
pressure)  of  light. 

Arrhenius  in  his  book  entitled  "Worlds  in  the  Making"  very  lucidly 
sets  forth  the  pros  and  cons  of  the  idea  of  panspermia  and  his  general  con- 
clusion is  that  the  interplanetary  dissemination  of  bacterial  spores  is 
within  the  possible. 

Schwarzschild  has  determined  mathematically  that  spherical  particles 
measuring  o.i/i  in  diameter  are  most  markedly  affected  by  the  radiation 
pressure  of  sunlight.  As  is  well  known  to  bacteriologists,  the  endospores 
of  bacteria  do  not  measure  more  than  from  0.2/4  to  0.3/1  in  diameter, 
even  less  in  the  dry  state.  The  compound  microscope  reveals  living 
plasmic  particles  which  are  less  than  0.025  m  diameter.  The  ultramicro- 
scope  enables  us  to  visualize  particles  of  gold  and  of  other  minerals  in 
colloidal  suspension,  which  particles  are  said  to  approximate  molecular 
dimensions. 

The  filterable  viruses  of  yellow  fever,  rabies,  smallpox,  the  mosaic 
disease  of  tobacco,  foot  and  mouth  disease  of  cattle,  and  others,  are  even 
smaller  than  the  endospores.  For  example  the  cytoryctes  (Cytorhyctes 
vaccinece)  which  is  presumed  to  be  the  primary  cause  of  smallpox,  meas- 
ures from  0.5/4  to  less  than  o.2jj,  in  diameter  when  suspended  in  liquids. 
In  the  dry  state  the  smaller  specimens  would  measure  less  than  0.065/4 
in  diameter. 

Very  naturally  the  question  arises  how  long  a  period  of  time  is  re- 
quired for  these  pangerms  to  be  carried  from  one  planet  to  another. 
It  has  been  determined  mathematically  that  a  germ  from  the  earth  would 
reach  Mars  in  twenty  days,  Jupiter  in  eighty  days,  Neptune  in  fourteen 
months  and  our  nearest  solar  system  (Alpha  in  Centauri)  in  9000  years. 
Can  spores  and  low  organisms  survive  these  periods  and  the  conditions 
known  to  exist  in  interstellar  space?  The  answer  is  in  the  main  in  the 


THE    ORIGIN   OF  BACTERIA   AND    OTHER  MICRO-ORGANISMS         2$ 

affirmative.  Spores  of  higher  fungi  and  of  lichens  have  survived  herba- 
rium conditions  for  many  years,  according  to  some  authorities,  for  nearly 
IQO  years.  Endospores  and  some  of  the  filterable  viruses  are  known  to 
survive  for  several  years  and  longer,  in  cloth,  in  dry  dirt  and  in  paper. 
The  microscopical  examination  of  the  cloth  of  Egyptian  mummies  showed 
the  presence  of  numerous  spores  of  some  mold,  also  endospores  of  bacteria, 
and  bacteria,  apparently  in  good  morphological  condition.  Some  of  these 
apparently  developed  when  introduced  into  gelatine  media,  though  it  was 
also  evident  that  most  of  them  had  lost  the  power  to  germinate  or  to 
septate.  In  interstellar  space  the  conditions  are  almost  ideal  for  the 
preservation  of  the  life  of  micro-organisms  in  the  resting  stage.  There  is 
absence  of  oxygen  of  moisture  and  the  temperature  is  very  low  (perhaps 
less  than  —220°  F.)'.  It  is  true  they  are  exposed  to  light,  more  especially 
the  chemically  active  ultraviolet  rays,  but  even  these  are  checked  in  their 
destructive  action  on  life  because  of  the  absence  of  warmth,  moisture  and 
of  atmospheric  oxygen.  There  is,  therefore,  no  plausible  reason  why  the 
minute  particles  detached  from  the  meteoric  mass  should  not  be  carried 
through  cosmic  space  by  the  radiation  pressure  of  sunlight.  This  radiation 
pressure  would,  however,  act  only  in  one  direction  within  our  solar  system, 
namely,  in  the  direction  away  from  the  sun.  This  force  could  therefore 
transport  germs  to  the  earth  from  Mercury,  Venus,  Moon  and  such 
meteoric  and  other  heavenly  bodies  which  move  between  the  earth  and  the 
sun.  None  could  reach  us  from  Mars,  Neptune  or  Jupiter,  unless  perhaps 
through  the  radiation  pressure  of  light  reflected  from  these  planets  but  since 
only  an  infinitely  small  amount  of  reflected  light  reaches  us  from  these 
sources  the  likelihood  of  this  carrying  life  to  the  earth  is  correspondingly 
slight.  However,  the  radiation  pressure  from  a  neighboring  sun  might 
carry  germs  to  the  planets  of  our  system. 

Very  naturally  the  question  arises  how  may  spores  and  other  similarly 
small  organisms  get  away  from  the  so-called  force  of  gravity  which  holds  all 
substances  of  a  planet  together  and  attracts  matter  in  space  to  its  surface. 
Air  currents  could  readily  carry  these  particles  to  the  upper  air  zone  but 
could  not  project  them  beyond.  Arrhenius  assumes  that  the  electrical 
currents  of  the  earth,  more  especially  the  negative  currents  of  the  north 
pole,  are  more  than  sufficiently  strong  to  carry  very  small  particles  against 
the  force  of  gravity  and  it  is  suggested  that  these  electrical  currents  are 
constantly  scattering  innumerable  spores  and  germs  into  cosmic  space. 
These  can  never  reach  the  sun  because  in  time,  in  their  flight  toward  that 
body,  the  radiation  pressure  of  light  will  arrest  them  and  even  turn  them 
back  upon  their  own  path. 

The  following  is  quoted  from  the  book  by  Arrhenius : 

"In  the  vicinity  of  solar  bodies  seeds  would  be  checked  in  their  cosmic 


26  PHARMACEUTICAL  BACTERIOLOGY 

flight  by  the  radiation  pressure  of  sunlight  and  tend  to  accumulate  in 
great  numbers.  The  planets  rotating  about  the  suns  would  thus  be  likely 
to  receive  such  seeds,  more  especially  those  planets  which  were  somewhat 
more  remote  from  the  sun.  In  these  positions  the  seeds  would  also  have 
lost  much  of  their  original  speed  and  would  in  all  probability  not  become 
excessively  heated,  probably  less  than  100°  C.,  in  their  motion  through  the 
atmosphere. 

"  In  the  vicinity  of  the  suns,  the  intercosmic  seeds  now  entering  upon 
their  return  journey  to  the  planets  would  meet  with  particles  whose  weight 
is  somewhat  less  than  the  repelling  force  of  the  radiation  pressure  and 
which  for  that  reason  have  begun  the  return  journey  to  the  sun.  Just 
like  the  seeds,  these  small  particles  tend  to  accumulate  in  the  vicinity  of 
the  suns. 

"These  very  small  seeds  and  the  yet  smaller  particles  clinging  to  them 
such  as  spores  and  bacteria,  would  be  more  likely  to  reach  the  planets 
nearer  the  sun. 

,  "  In  this  manner  life  may  have  been  carried  from  planet  to  planet,  from 
solar  system  to  solar  system,  throughout  the  ages.  But,  as  in  the  case  of 
the  billions  of  pollen  grains  which  escape  from  a  single  oak,  and  which 
are  distributed  by  the  air  currents,  perhaps  only  one  will  give  rise  to  a 
new  tree;  so  likewise  of  the  billions  and  trillions  of  germs  which  wander 
about  in  cosmic  space,  only  one  may  ever  reach  a  planet  where  it  may  de- 
velop and  give  rise  to  many  new  forms  of  life." 

According  to  the  above  idea  of  panspermia  all  of  the  organisms  in  the 
entire  universe  are  related  and  consist  of  cells  which  are  built  up  through 
chemical  combinations  of  carbon,  hydrogen,  oxygen  and  nitrogen.  The 
supposition  that  there  may  be  worlds  in  which,  for  example,  living  organ- 
isms are  formed  from  chemical  combinations  in  which  carbon  might  be 
displaced  by  silicon  or  titanium,  is  highly  improbable.  Life  upon  other 
inhabited  planets  is  in  all  probability  similar  to  that  upon  the  earth. 

It  is  generally  known  that  several  planets  of  our  solar  system  are  closely 
similar  to  that  of  the  earth,  as  Venus  and  Mars.  Mercury,  like  the  moon, 
turns  one  side  to  the  sun  at  all  times  and  hence  the  sides  have  a  constant 
temperature,  one  side  hot  and  the  other  cold,  one  side  in  constant 
darkness  and  the  other  constantly  illumined. 

Even  admitting  that  the  idea  of  panspermia  is  well  founded  and  that 
very  minute  forms  of  life  can  be  transmitted  from  one  planet  to  another,  the 
question  of  the  origin  of  life  still  remains  unanswered.  We  have  merely 
pushed  the  problem  into  a  dark  corner.  By  relegating  the  matter  to  one 
or  many  distant  planets  we  have  not  thereby  escaped  the  responsibility  of 
the  final  scientific  proof  and  unimpeachable  explanation  of  the  origin  of  life. 

Bacteriologists  have  from  time  to  time  commented  upon  the  fact 


THE    ORIGIN   OF   BACTERIA   AND    OTHER   MICRO-ORGANISMS          27 

that  bacteria  are  morphologically  as  well  as  physiologically  essentially 
different  from  other  living  things  upon  the  earth,  more  especially  those 
species  which  form  endospores.  As  already  suggested,  bacteria  apparently 
have  no  phylogenetic  relationship  to  any  of  higher  forms  of  planet  or 
animal  life,  nor  yet  to  any  of  the  recognized  protozoa  or  protophyta. 
May  it  not  be  possible,  for  example,  that  the  xerophytic  and  anaeroJbic_ 
pathogens,  such  as  the  Bacillus  tetani,  B.  botulinus,  and  B.  cedematis 
maligni  have  reached  us  from  the  moon,  where  the  meteorological  conditions 
are  suitable  for  the  existence  of  this  type  of  organism.  May  it  not  even 
be  possible  that  these  pathogens  of  comparatively  high  specific  gravity  and 
endowed  with  extreme  temperature  resistance  were  the  chief  actors  in 
the  final  lunar  struggle  for  the  survival  of  higher  life.  As  the  moon  was 
bombarded  by  planetary  bodies  most,  if  not  all,  of  the  higher  organisms 
were  no  doubt  killed  by  the  shocks  of  impact  and  the  heat  generated. 
Such  higher  forms  as  survived  were  unable  to  continue  when  the  life 
sustaining  atmosphere  and  moisture  become  more  and  more  reduced, 
thus  favoring  more  and  more  those  .lowly  organized  living  structures 
which  could  thrive  in  this  rarefied  atmosphere  and  dry  condition. 

As  suggested  the  comparatively  large  and  highly  refractive  toxigenic 
spore  forming  bacilli  may  have  reached  us  from  the  moon.  The  smaller 
plasmodia  and  very  small  non-spore-forming  bacilli  may  have  come  to  us 
from  Mars  and  Venus,  and  perhaps  also  from  Mercury.  The  temperature 
on  Venus  is  higher  than  it  is  on  the  earth  whereas  the  mean  temperature 
on  Mars  is  lower,  even  though  the  polar  snows  of  that  planet  occasionally 
disappear  entirely  during  some  seasons,  which  never  happens  on  the  earth. 
These  differences  in  planetary  temperatures  suggest  that  the  germs  of 
yellow  fever,  of  amebic  dysentery,  of  the  African  sleeping  sickness,  of 
leprosy  and  perhaps  also  of  malaria,  may  have  come  from  Venus ;  whereas 
the  germs  of  rabies,  of  la  grippe,  of  whooping  cough,  of  scurvy,  of  the 
plague  and  perhaps  also  of  smallpox,  may  have  come  from  Mars. 

4.  The  Theory  of  Universal  Evolution. — The  belief  in  the  genetic 
or  evolutional  relationship  of  all  things  in  the  entire  Universe,  is  of  ex- 
treme antiquity.  According  to  an  ancient  Hindoo  myth,  at  the  beginning 
of  things  there  existed  a  mundane  or  cosmic  egg  or  germ  from  which  all 
animate  as  well  as  inanimate  things  successively  emerged.  The  scholars 
of  Athens,  of  Rome  and  of  Alexandria  held  similar  ideas.  Later,  the  idea 
became  more  definitely  formulated  by  such  master  minds  as  Huxleyr 
Spencer  and  others.  The  same  idea  is  further  developed  and  matured  by 
the  teachings  of  the  physicists  and  now  we  find  ourselves  compelled  to  go 
back  in  our  study  of  evolution  and  bring  with  us  all  matter,  organic  and 
inorganic,  living  and  dead,  atom,  molecule  and  compound. 

Biological  science  has  convinced  us  that  death  and  decay  are  essential 


28  PHARMACEUTICAL  BACTERIOLOGY 

to|the  renewal  of  life  and  to  the  advance  or  evolution  of  living  things 
and  now  the  physicist  presents  a  similar  idea  applied  to  the  atom  and  to 
the  molecule.  The  atom  is  no  longer  considered  to  be  the  ultimate  unit  of 
matter,  unchangeable,  indivisible  and  undestructible.  The  atom  is 
rather  a  miniature  universe  consisting  of  a  definitely  known  electric  charge 
composed  of  a  positive  nucleus  about  which  revolve  the  negative  electrons 
in  definite  orbits.  The  newer  teaching  of  physics  is  to  the  effect  that  the 
only  difference  between  hydrogen  and  oxygen,  for  example,  is  that  the 
oxygen  atom  has  16  times  more  negative  electrons  grouped  about  the 
positive  nucleus.  The  radium  atom  differs  only  in  the  greater  number  of 
electrons.  The  molecule,  no  matter  how  simple  or  how  complex,  is 
nothing  more  than  a  grouping  of  electrons.  If  this  be  anywhere  near  the 
actual  facts,  and  much  of  the  experimental  evidence  is  confirmatory,  we 
at  once  obtain  a  new  significance  of  the  periodic  grouping  of  the  elements 
(as  given  by  Mendeleeff  and  Lothar  Meyer)  and  the  relationship  of  mole- 
cules. Life  is  thus  resolved  into  electronic  groupings  and  electronic  action. 
Life  is  thus  nothing  more  nor  less  than  the  manifestation  of  properties  pecul- 
iar to  certain  definite  electronic  groupings  as  represented  by  certain  molecu- 
les. Living  things  appeared  at  the  precise  moment  when  the  electronic  act- 
ivity represented  by  the  appropriate  atomic  grouping  in  the  molecules  was 
such  that  it  could  be  designated  by  the  term  life.  When  man  shall  have  ac- 
quired the  ability  to  imitate  this  grouping  he  will  then  have  developed 
a  living  thing.  Life  is  thus  nothing  more  nor  less  than  a  problem  in 
physical  science. 

The  physicist  tells  us  that  the  disintegrating  radium  atom  breaks  up 
into  free  electrons  manifest  as  beta  rays,  and  perhaps  into  free  or  compara- 
tively free  positive  nuclei  and  that  some  of  these  disrupted  atomic  con- 
stituents are  again  rearranged  or  recombined  into  new  atoms  of  helium 
represented  by  the  alpha  rays.  What  starts  the  radium  atom  on  its  un- 
alterable explosive  disintegrating  course?  It  is  assumed  that  under  cer- 
tain conditions,  perhaps,  occasioned  by  the  change  in  the  orbital  position 
and  rotation  of  the  negative  electrons  about  the  positive  nucleus,  due 
to  the  mutual  repulsion  of  the  electrons,  there  is  a  sudden  explosion  of  the 
atom  and  the  electrons  are  shot  off  into  space.  These  free  electrons 
moving  at  a  speed  approximating  that  of  light  and  obeying  the  laws  °f 
"falling"  bodies,  may  again  reform  in  orbits  about  a  free  or  comparatively 
free  positive  nucleus,  perhaps  a  nucleus  from  which  they  have  just  been 
shot  forth,  but  the  new  atomic  orbital  grouping  of  the  electrons  is  now 
entirely  different,  resulting  in  the  formation  of  a  new  atom.  Only  the 
dying  (disintegrating)  atoms  make  the  formation  of  new  atoms  possible, 
for  as  far  as  we  know  at  the  present  time,  no  new  matter  is  added  to  the 
universe  nor  is  there  any  destroyed  (annihilated)  or  taken  therefrom. 


THE   ORIGIN   OF  BACTERIA   AND    OTHER   MICRO-ORGANISMS         2Q 

It  is  no  pathological  stretch  of  the  imagination  to  suppose  that  the 
macrocosm  (as  represented  by  a  stellar  system)  is  mirrored  in  the  micro- 
cosm (as  represented  by  the  atom  and  the  molecule).  There  can 
be  little  doubt  that  old  stellar  systems  disintegrate  and  new  ones  form. 
Nebulae,  comets  and  stellar  dust  are  not  newly  created  substances  as  is 
generally  taught,  but  rather  newly  reformed  or  rearranged  substances^jre- 
sulting  from  explosively  disintegrated  stellar  systems,  in  similitude  to  dis- 
integrating atoms  (radium,  thorium,  polonium,  lead,  etc.). 

Bacteriology  is  the  newest  of  the  sciences,  dating  back  to  about  1875. 
And  since  then  this  science  has  made  a  series  of  explosive  advances.  It  is 
generally  taught  that  bacteria  are  plants  and  it  has  been  customary  to  class 
them  with  the  fungi,  and  some  scientists  have  even  suggested  that  they  are 
somatically  reduced  or  degenerate  algae.  There  is,  however,  no  good 
reason  for  assuming  that  they  are  degenerate  algae  nor  are  we  justified  in 
designating  them  as  plants.  We  are  justified  in  stating  that  the  various 
groups  of  bacteria  are  phylogenetically  related  and  that  they,  in  all  proba- 
bility, had  a  polyphyletic  origin  in  many  different  areas  of  the  earth's 
surface,  or  mayhap  upon  other  planets  as  has  been  explained  above. 
We  are  at  the  present  time  not  in  a  position  to  state  definitely  whether  or 
not  any  of  the  higher  fungi  are  phylogenetically  derived  from  any  of  the 
groups  of  bacteria.  The  Leptothrix  and  Streptothrix  groups  are  perhaps 
of  bacterial  origin.  We  are  amply  justified  in  saying  that  bacteria  were 
among  the  first,  if  not  the  first,  living  things  which  made  their  appearance 
upon  an  originally  lifeless  earth.  We  know  that  the  large  group  of  nitri- 
fying bacteria  will  grow  in  a  medium  composed  of  water  to  which  is  added 
o.i  per  cent,  each  of  ammonium  sulphate,  potassium  phosphate  and 
magnesium  carbonate,  a  medium  wholly  free  from  sugar  and  nitrogenous 
compounds  containing  only  such  ingredients  as  existed  on  our  plant  prior 
to  the  development  of  living  things.  Out  of  these  substances  the  nitrifiers 
formed  (in  the  presence  of  air)  ammonia,  sugar,  fatty  acids  and  proteins, 
which  substances  in  turn  serve  as  media  for  the  development  of  other 
bacteria  and  of  higher  organisms.  This  statement  will  serve  to  indicate 
the  potentialities  of  this  group  of  bacteria  in  the  way  of  assimilating  dead 
inorganic  matter  and  converting  it  into  or  utilizing  it  as  a  life-sustaining 
pabulum.  We  are,  however,  still  confronted  with  the  problem  of  the 
source  of  the  life,  the  life  principle  or  whatever  it  maybe  styled,  which  made 
it  possible  for  bits  of  organic  matter  to  utilize  these  dead  inorganic  materials 
for  the  purpose  of  maintaining  life  and  as  the  building  material  of  a 
living  substance,  for  life  activities  imply  the  existence  of  a  living  organic 
substance. 

It  may  be  recalled  that  some  years  ago  Huxley  thought  he  had  dis- 
covered the  primal  living  substance  in  the  slime  taken  from  the  ocean's  bed 


30  PHARMACEUTICAL  BACTERIOLOGY 

and  which  substance  he  named  Bathybius  Hceckelii.  This  supposed  deep 
sea  life  was  found  to  consist  largely  of  colloidal  deposits.  The 
theory  of  spontaneous  generation  is  fully  disproven  though  considerable 
effort  has  been  expended  in  an  attempt  to  form  a  living  substance  in  the 
laboratory.  Recently  Dr.  Burke  claimed  to  have  succeeded  in  instilling 
life  into  gelatine  solutions  by  means  of  radium,  but  this  proved  to  be  an 
error. 

Biitchli's  "artificial  protoplasm"  was  once  of  considerable  laboratory 
interest,  but  it  was  not  intended  that  this  substance  (an  emulsion  of  oil) 
should  be  considered  as  being  endowed  with  life.  The  artificial  continua- 
tion of  tissue  growth  according  to  the  laboratory  methods  of  Dr.  Carel  and 
others,  of  course,  has  no  bearing  on  the  creation  of  living  cells  by  labora- 
tory methods. 

According  to  the  theory  of  universal  evolution,  the  origin  of  bacteria 
was  simply  an  incident  in  the  series  of  events  (electronic)  which  resulted  in 
the  development  of  the  particular  substances  which  occur  in  that  particular 
group  of  organisms,  and  the  theory  could  be  appended  to  the  preceding 
one  or  to  the  one  which  follows,  since  there  is  nothing  in  the  concept  of 
universal  evolution  which  conflicts  with  the  ideas  therein  presented. 
4       5.  The  Colloid  Theory. — The  word  colloid  is  derived  from  the  latin 
collo  (glue)  and  was  applied  by  Thomas  Graham,  the  father  of  colloidal 
chemistry,  to  non-cry stalizable  substances  of  low  diffusibility  and  gener- 
ally of  great  viscosity,  and  other  characteristic  properties.     Graham's 
experiments  on  dialysis  and  the  general  properties  of  colloids,  were  read 
before  the  Royal  Society  of  London  in  1861,  from  which  it  becomes  evident 
that  colloidal  chemistry  is  the  youngest  of  the  modern  sciences.     The  still 
more  recent  investigations  in  the  field  of  colloids  has  been  fruitful  in  sug- 
gestions and  theorizations  regarding  the  nature  of  living  organic  substances. 
In  order  to  comprehend  the  basic  principles  of  the  colloid  theory  of  living 
matter,  it  is  necessary  to  set  forth  the  fundamentals  of  colloids.     Gelatine 
may  be  taken  as  a  type  of  colloidal  matter.     Colloids  never  crystallize, 
they  will  not  pass  through  animal  membranes.     A  colloid  dissolved  in 
water  is  designated  a  "sol,"  and  as  water  is  gradually  abstracted  the  sol 
changes  into  a  "gel."     A  sol  is  a  colloid  system  consisting  of  two  phases, 
the  continuous  phase  called  the  dispersing  medium  or  dissolving  medium, 
and  the  disperse  phase  or  the  colloidal  particles  in  solution.     Two  classes 
of  colloids  are  of  special  importance  to  biology,  namely  the  emulsoids  and 
the  suspensoids.     The  emulsoids  are  viscous,  gelatinize  readily  and  are 
not  easily  coagulated  by  electrolytes.     Among  the  important  emulsoids 
are  gelatine,  agar,  albumin,  histons,  and  other  proteins.     The  suspen- 
soids do  not  gelatinize,  they  are  not  viscous,  but  are  readily  precipitated 
by  electrolytes.     Among  the  suspensoids  are  the  sols  of  metals  generally, 


THE    ORIGIN   OF  BACTERIA   AND    OTHER   MICRO-ORGANISMS         31 

and  of  some  of  the  dyes.  It  may  also  be  stated  that  the  colloid  state  is  not 
restricted  to  any  one  group  or  even  many  groups  of  matter,  rather  it  is 
universal.  Any  and  all  substances  may  be  converted  into  the  colloid 
state,  including  the  chemical  salts.  In  general  it  is  however  true  that 
substances  with  small  molecules  prefer  the  crystalloidal  state  or  form, 
whereas  the  substances  consisting  of  large  molecules  prefer  the  colloidal ~ 
"'  statl^  Matter  in  the  colloidal  state  consists  of  minute  molecular  aggre- 
grates  (not  chemical  combinations)  varying  in  size  from  macroscopic  ,that 
is  large  enough  to  be  visualized  by  the  naked  eye  (at  least  when  the  light 
rays  are  properly  adjusted)  down  to  aggregates  which  are  so  small  that 
they  may  not  even  be  visualized  by  means  of  the  ultra-miscoscope. 
Another  property  of  colloidal  matter  is  the  manifestation  of  the  very 
striking  Brownian  motion.  In  a  general  way  colloidal  particles  are  de- 
cidedly opposed  to  chemical  combinations.  In  fact  the  study  of  colloids 
lies  almost  wholly  in  the  realm  of  physics,  rather  than  chemistry. 

The  recent  investigations  in  bio-chemistry  and  colloidal  chemistry 
have  shown  that  life  processes  take  place  in  colloid  systems.  Life,  death 
and  decay,  and  renewed  life,  form  one  continuous  kalaidoscopic  colloidal 
transformation  series,  in  which  the  organic  molecules,  water  and  enzymes 
play  the  leading  roles.  The  secrets  of  living  matter  are  bound  up  in 
colloids.  Not  the  coarser  colloids  recognizable  microscopically  or  even 
ultra-microscopically,  but  in  those  protein  and  histon  colloids  which 
abound  near  the  border  line  of  the  true  (molecular)  solutions.  The 
living  basic  substance  in  which  all  of  the  life  processes  take  place,  that  is 
the  plasm  (stroma),  is  a  protein  emulsoid  in  water.  It  is  itself  a  dialyzing 
substance  which  will  take  up  and  will  allow  to  enter  and  pass  the  smallest 
colloidal  particles  only.  It  is  a  labilely  stable  substance.  That  is,  it  is 
chemically  stable  under  certain  conditions  of  light,  temperature  and  en- 
vironment. The  plasmic  granula  which  may  be  seen  under  the  high  power 
of  the  compound  microscope,  or  visualized  under  the  ultra-microscope,  and 
all  of  the  known  formed  cell  constituents,  inclusive  of  nuclear  matter,  are 
precipitation  and  coagulation  products  of  plasmic  activity.  The  cell- 
walls,  the  starch  granules,  crystals  of  calcium  oxalate,  etc.,  of  plant  cells, 
represent  refuse  material,  rejected  by  the  living  plasm.  Gradually,  the 
plasm  is  unable  to  find  sufficient  dumping  space  for  the  refuse,  the  cell- 
wall  growing  thicker  and  thicker,  the  cell  lumen  smaller  and  smaller,  and 
finally  the  plasm  dies  upon  the  heap  of  refuse  of  its  own  building.  It  is 
true,  that  the  starch  and  other  plasmic  rejecta  may  again  be  utilized  in 
subsequent  growth  processes,  and  the  cell-wall  serves  as  a  protection 
against  the  loss  of  moisture  and  also  assists  in  colloidal  filtration  so  essential 
to  the  life  of  the  plasm.  The  nucleus  of  the  cell  is  nothing  more  nor  less 
than  a  series  of  colloidal  coagulation  changes,  of  colloid  coagula  tern- 


32  PHARMACEUTICAL  BACTERIOLOGY 

porarily  thrown  off  by  the  plasmic  base  or  stroma,  a  temporary  resting 
condition  of  a  portion  of  the  plasmic  stroma,  to  take  on  renewed  activity 
as  soon  as  the  conditions  shall  have  become  favorable.  The  nucleus  and 
all  that  pertains  thereto  may  be  likened  to  the  cell  plastids  and  other 
living  inclusions  of  the  cell,  in  so  far  as  they  are  plasmic  rejecta  or  storage 
matter,  temporarily  set  aside  to  be  activated  by  the  plasmic  base  when  the 
conditions  are,  for  example,  suitable  for  cell  division  to  take  place.  That 
plasm  is  king,  and  not  the  nucleus,  is  proven  by  the  fact  that  enucleated 
cells  may  be  induced  to  grow  and  septate,  whereas  nuclei  separated  from 
the  cell  plasm  will  not  divide  and  develop  into  new  cells.  It  is  highly 
probable  that  the  substances  which  a  cell  deposits  in  the  nucleus,  leaves 
the  vitality  of  the  plasm  very  much  weakened  and  unless  the  plasm  makes 
use  of  this  stored  nuclear  substance,  it  will,  as  a  general  rule,  not  be  able 
to  survive  for  long  periods,  and  under  usual  or  natural  conditions  finds 
itself  incapacitated  for  cell  division,  or  even  for  cell  growth.  The  sphaero- 
cytes  (nucleated  living  cell  inclusions  common  in  fruits)  are  apparently 
extra  deposits  of  the  cell  plasm  which  have  the  power  to  continue  the  life 
of  the  cell  for  a  time,  thus  enabling  fruits  to  remain  alive  for  many  months 
after  they  have  ripened  and  separated  from  the  mother  plant.  If  the 
living  cell  could  dispose  of  the  coagulation  and  precipitation  products 
as  rapidly  as  they  are  formed  then  the  cell  would  continue  to  live  indefi- 
nitely. The  Ameba  does  this  to  all  intents  and  purposes,  because  the  dis- 
persing medium  in  which  it  lives  (namely  water)  disperses  the  rejected 
products  at  once.  Death  among  the  multicellular  organisms  is  inevitable, 
unless  some  condition  develops  which  will  enable  the  organism  to  get  rid 
of  the  coagulation  and  precipitation  products.  •  In  this  lies  the  solution  to 
the  problem  of  eternal  life  and  eternal  youth.  We  may  indeed  reduce  the 
precipitation  changes  to  a  minimum,  by  reducing  the  plasmic  activities  to 
a  minimum.  Such  conditions  exist  in  spores,  in  seeds,  and  in  the  resting 
stages  of  various  organisms. 

From  the  foregoing  it  must  be  apparent  that  life,  as  we  know  it,  could 
not  have  come  into  existence  until  certain  highly  complex  molecules  had 
developed,  which  complex  molecules  must  be  in  the  proper  colloidal  com- 
minution and  dispersed  in  water  and  associated  with  other  colloidal 
particles,  including  minerals,  as  sulphur  and  phosphorus.  Water  takes 
no  active  part  in  the  life  processes,  although  it  is  the  absolutely  essential 
dispersing  medium  for  the  plasmic  base  or  stroma.  Unless  plasm  is 
actually  immersed  in  water  from  which  it  can  draw  unreservedly,  it  must 
constantly  have  water  brought  to  it,  otherwise  it  cannot  continue  in  the 
living  state.  We  may  make  certain  statements  as  to  the  properties  of  the 
living  stroma  in  which  all  life  activities  take  place  and  from  which  all 
plasmic  formations  are  derived. 


THE   ORIGIN   OF  BACTERIA  AND    OTHER  MICRO-ORGANISMS         33 

1.  It  is  a  true  colloid  phase  in  which  water  is  the  dispersing  medium, 
and  in  which  the  dispersoids  are  complex  protein  molecules  in  aggregates 
so  small  as  to  be  wholly  invisible  by  any  of  the  optical  aids  to  vision. 

2.  It  is  very  slightly  permeable  to  water  and  is  even  less  permeable 
to  the  solutes  which  may  be  in  the  water. 

3.  It  is  labilely  stabile  within  certain  conditions,  as  water  supply, 
temperature,  and  food  supply.     If  the  conditions  become  excessive  the 
precipitation  and  coagulation  changes  are  such  as  to  destroy  the  disperse 
phase  peculiar  to  a  living  substance. 

4.  In  brief,  the  plasmic  stroma  manifests  properties  of  a  colloidal 
filter,  in  which  the  pores  are  so  small  as  to  permit  entrance  and  passage 
to  the  very  smallest  colloidal  particles  only. 

It  may  be  assumed  that  life  came  into  existence  upon  the  earth  at  the 
precise  moment  when  the  particular  proteid  molecules  appeared  in  water, 
which  in  the  presence  of  certain  other  colloidal  suspensions,  formed  perhaps 
eons  earlier,  together  with  a  certain  temperature  and  other  environmental 
conditions,  gave  rise  to  the  coagulation  change  in  the  disperse  phase  to 
which  we  have  come  to  ascribe  a  living  state.  The  much  discussed 
" spark  of  life"  is  thus  nothing  more  nor  less  than  the  coming  into  being 
of  the  particular  colloid  substance  (protein)  which  was  essential  to  bring 
about  the  particular  physical  changes  to  which  we  ascribe  life.  Life 
is  thus  not  a  chemical  change  although  the  changes  in  matter  which  are 
essential  to  give  rise  to  those. substances  which  may  be  used  as  food  by  the 
living  substance,  or  rather  which  are  essential  for  the  purpose  of  main- 
taining those  constantly  varying  physical  (colloidal)  conditions  which  we 
designate  as  manifestations  of  life,  are  chemical.  Enzymes  in  particular, 
play  a  very  important  part  in  those  chemical  and  physical  changes  so 
essential  to  higher  life. 

The  artificial  production  of  life  in  the  laboratory  is  not  so  much  a 
matter  of  providing  a  suitable  environment  to  which  organic  matter  must 
be  exposed,  as  it  is  a  matter  of  finding  a  suitable  colloidal  filter.  Should 
we  prepare  a  colloidal  filter  having  the  filtering  qualities  of  the  plasmic 
stroma,  we  might  then  be  prepared  to  begin  upon  a  series  of  laboratory 
experiments  with  a  view  to  producing  a  living  colloid  substance.  Other 
factors  altogether  too  numerous  to  mention  come  into  play  also.  The 
above  is  a  mere  outline  of  the  present  concept  of  the  colloidal  nature  and 
origin  of  living  substances. 

Many  other  theories  have  been  presented  but  none  of  them  have  any- 
thing new  or  essentially  different  from  the  ideas  above  outlined.  Among 
the  investigators  who  have  given  the  subject  considerable  thought  may  be 
mentioned  Osborne  who  dwells  at  considerable  length  on  the  probable  con- 
ditions on  the  earth's  surface  during  the  geologic  ages.  He  is  of  the 


34  PHARMACEUTICAL  BACTERIOLOGY 

opinion  that  the  prototrophic  group  of  bacteria  were  the  first  living 
things  upon  the  earth's  surface.  Troland  is  of  the  opinion  that  enzymes 
played  the  leading  part  in  the  creation  of  life.  The  source  of  the  first 
enzyme  (protenzyme)  is  however  not  known.  If  all  enzymes  now  known 
are  of  living  origin  then  we  must  assume  that  the  protenzyme  came  into 
existence  before  the  protoplasm  (first  plasm).  Thus  the  Troland  sugges- 
tion is  really  of  no  help  in  explaining  the  orgin  of  life.  There  is  however 
no  reason  why  we  should  not  assume  that  protenzyme  and  protoplasm 
developed  at  one  and  the  same  time.  Allen  held  that  the  conditions  which 
maintain  life  are  also  conducive  to  the  creation  of  life,  and  should  by  chance 
all  life  on  the  earth's  surface  at  the  present  time  be  destroyed,  a  new 
cycle  of  life  would  begin  forthwith.  Pflliger  suggests  that  there  is  a  funda- 
mental difference  between  a  living  protein  and  a  dead  protein  and  that  the 
cyanogen  radical  is  the  distinctive  part  of  the  molecular  complex  of  living 
proteins.  Moore  was  one  of  the  first  to  give  serious  consideration  to  the 
importance  of  colloidal  changes  in  the  development  of  living  matter. 
While  none  of  the  investigators  have  yet  been  able  to  solve  the  problem  of 
life,  yet  the  numerous  propositions  which  have  been  made  from  time  to 
time,  and  the  yet  larger  number  of  theories  which  will  be  offered  in  the  near 
future,  are  indications  that  the  trend  of  science  is  in  the  same  direction 
and  it  is  but  reasonable  to  expect  that  some  one  will  in  the  not  very  distant 
future  find  the  solution. 


CHAPTER    IV 

GENERAL  MORPHOLOGY  AND  PHYSIOLOGY 
i.  INTRODUCTION 

Microbiology  in  the  true  and  comprehensive  sense,  is  the  science 
which  treats  of  microscopic  organisms,  micro-organisms  or  microbes, 
vegetable  as  well  as  animal.  It,  therefore,  comprises  a  study  of  bac- 
teria, of  yeasts,  of  the  lower  molds,  of  the  lower  algae,  of  protozoa,  of 
flagellata,  of  ciliata,  and  of  other  low  forms  of  plant  and  animal  life. 
Applied  in  a  more  limited  sense,  the  term  has  more  recently  come  into 
use  in  place  of  the  term  bacteriology.  The  latter  word  is  derived  from 
bacterium  (from  the  Greek,  bactros),  meaning  a  small  rod  because  the 
earlier  students  assumed  that  all  microbes  were  rod  shaped,  which  is  not 
the  case,  an  d  the  Greek  word  logos  meaning  discourse.  Microbiology  (from 
'micros,  small,  bios,  life;  and  logos  discourse)  even  when  applied  in  the 
narrower  sense,  is  therefore  etymologically  more  nearly  correct  than  is 
the  word  bacteriology.  The  only  excuse  for  using  the  words  bacteria 
and  bacteriology  is  established  usage. 

By  microbes  or  microscopic  organisms  are  meant  those  living  units 
which  are  so  small  as  to  render  the  individual  unrecognizable  to  the  naked 
or  unaided  eye.  As  generally  understood  microbes  are  so  small  as  to 
require  the  use  of  a  good  compound  microscope  to  bring  the  individual  or 
individuals  to  view.  A  good  simple  lens  or  simple  microscope,  having  a 
magnifying  power  ranging  from  four  to  ten  diameters,  will  reveal  the 
position  in  space  of  some  of  the  larger  forms  of  microbes  but  their  detailed 
structure  is  not  recognizable  under  such  limited  magnification.  Microbes 
in  mass  are  generally  visible  to  the  naked  eye,  thus  we  recognize  the  blue 
green  mold  on  bread,  bacterial  membrane  on  potato  and  vinegar,  algae  in 
water  and  in  soil,  etc. 

Some  microbes,  presumably  belonging  to  the  group  of  so-called  bac- 
teria, and  perhaps  also  to  the  group  protozoa,  are  supposed  to  be  too  small 
to  be  seen,  even  under  the  highest  power  of  the  compound  microscope  and 
-are  spoken  of  as  " ultra-microscopic."  This  is,  however,  mere  theoretical 
assumption  as  no  one  has  thus  far  succeeded  in  demonstrating  the  exist- 
ence of  ultra-microscopic  organisms. 

Although  the  terms  microbe  and  microbiology  are  here  used  in  the  true 
broad  and  comprehensive  sense,  the  subject-matter  of  the  present  volume 
is  very  largely  devoted  to  a  brief  summarizing  discussion  of  those  microbes 

35 


36  PHARMACEUTICAL  BACTERIOLOGY 

or  micro-organisms,  concerned  in  human  economy,  as  those  having  to  do 
with  health  and  disease,  directly  and  indirectly,  thus  including  scientific 
(theoretical)  and  applied  bacteriology  in  the  older  sense  with  its  more 
modern  subdivisions  as  medical  bacteriology,  food  bacteriology,  pharma-  • 
ceutical  bacteriology,  dental  bacteriology,  soil  bacteriology,  veterinary 
bacteriology,  dairying  bacteriology,  poultry  bacteriology,  agricultural 
bacteriology,  etc.,  and  also  medical  and  sanitary  parasitology,  much  of 
pathology,  general  zymology  immunology  and  scientific  (theoretical) 
as  well  as  practical  or  applied,  serology ;  and  still  more  remotely  the  subject 
also  includes  the  fundamentals  of  public  health  and  hygiene,  sanitation 
and  preventive  medicine  in  general. 

There  are  certain  important  factors  not  directly  pertaining  to  the 
science  of  microbiology  as  above  outlined  which  nevertheless  have  more  or 
less  direct  bearing  upon  the  subject  and  which  must  be  touched  upon  for 
the  sake  of  completeness  and  of  a  fuller  understanding,  such  as  carriers  of 
disease,  secondary  causes  of  disease,  disinfectants  and  disinfection,  food 
preservatives,  etc. 

Bacterium  (plural,  bacteria)  is  a  misleading  term,  though  firmly  estab- 
lished in  general  usage.  Furthermore,  the  term  is  used  in  a  generic  sense, 
and  again  applied  to  the  group  of  organisms  as  a  whole.  This  causes 
confusion.  Therefore,  the  generic  term  Bacterium  is  now  abandoned 
and  the  term  Bacillus  is  used  to  include  all  of  the  micro-organisms  which 
are  rod-shaped  although  generic  sub-divisions  are  being  made  of  this  now 
very  large  group. 

Whereas  the  general  morphology  of  microbes  is  apparently  quite 
simple,  the  physiology  and  chemistry  is  extremely  complex,  and  as 
yet  not  fully  understood.  The  morphological  simplicity  is  no  doubt  only 
apparent,  and  not  real.  Perhaps,  with  the  greater  perfection  of  the 
compound  microscope,  we  may  discover  marked  structural  differences 
which  thus  far  have  escaped  our  notice. 

i.    Classification  of  Microbes 

Microbes  are  the  smallest  of  the  known  living  organisms.  It  is  wholly 
impossible  to  see  the  single  individual,  even  the  largest,  with  the  naked  eye. 
The  rod-shaped  microbes  (bacilli)  range  from  0.5/4  to  io/*  in  length.  Some 
are  so  minute  as  to  pass  through  the  pores  of  the  finest  clay  filters  (the 
cause  of  foot  and  mouth  disease).  To  study  them,  a  good  compound 
microscope  is  absolutely  necessary,  though,  as  stated  in  the  historical 
review  (Period  II),  Leeuwenhoek  and  others  observed  the  larger  forms 
under  the  simple  microscope. 

The  systematic  position  of  microbes  has  from  time  to  time  received 
much  attention.  The  great  majority  of  biologists  now  unhesitatingly  class 


GENERAL   MORPHOLOGY   AND   PHYSIOLOGY  37 

them  as  plants,  belonging  to  the  group  fungi.  It  cannot  be  denied,  how- 
ever, that  their  origin  (phylogeny)  is  still  shrouded  in  mystery.  Some  sug- 
gest that  they  are  derived  from  degenerate  algal  forms,  in  common  with 
most  of  the  fungi,  while  others  declare  that  they  in  all  probability  origi- 
nated as  microbes.  A  few  of  the  philosophical  biologists,  as  Ernst  Haeckel, 
place  them  in  a  separate  group,  the  Monera,  which  is  supposed  to  form  the 
connecting  link  between  plants  and  animals. 

Without  entering  into  lengthy  discussion,  we  shall,  in  conformity  with 
the  opinion  of  the  majority,  class  them  as  plants,  belonging  to  the  lowest 
of  the  group  fungi  (the  fungi  includes  rust,  smuts,  cup  fungi,  moulds,  spot 
fungi,  toad-stools,  etc.),  namely,  the  Schizomycetes  or  fission  fungi,  so- 
called  because  they  multiply  by  fission  or  division.  They  are  related  to 
the  yeasts,  though  somewhat  lower  in  the  scale  of  evolution.  They 
are  single-celled,  each  cell  forming  a  complete  living  unit,  though  the 
several  units  may  be  variously  arranged  into  chains  or  clusters,  or  groups 
known  as  zoogloea. 

The  scientific  grouping  of  microbes  is  as  yet  very  unsatisfactory  because 
so  little  is  known  of  their  ultimate  morphology,  their  physiology  and 
chemistry.  Some  have  attempted  to  classify  them  as  to  form,  others  as 
to  occurrence,  as  to  action,  etc.  Thus,  we  have: 

a.  Micrococci  or  Coccaceae. — Globular  or  non-elongated  microbes. 

b.  Bacilli    or  Bacteriaceae. — Cells  more    or    less  elongated.     Rod- 
shaped  microbes. 

c.  Spirillae  or  Spirillaceae. — Cells  elongated  and  more  or  less  spirally 
twisted.     Or,  we  may  have : 

a.  Bacteria  of  earth. 

b.  Bacteria  of  air. 

c.  Bacteria  of  water. 
Or,  again: 

a.  Chromogenic. 

b.  Zymogenic. 

c.  Pathogenic,  etc. 

These  artificial  groupings  could  be  extended  indefinitely,  but  such  sys- 
tems of  classification  would  be  as  unsatisfactory  as  they  are  unscientific. 
The  best  system  makes  use  of  all  of  the  known  facts  of  bacteriology. 
Several  such  systems  have  been  proposed  from  time  to  time,  but  the  new 
discoveries  along  bacteriological  lines  makes  it  necessary  to  change  them 
in  the  course  of  two  or  three  years.  Migula,  Fischer,  Eisenberg  and  others 
have  proposed  general  systems,  and  a  host  of  investigators  have  submitted 
more  limited  group  systems.  The  following  classification  will  serve  to 
convey  some  idea  as  to  the  structural  characteristics  of  the  more  impor- 
tant groups : 


38  PHARMACEUTICAL  BACTERIOLOGY 

i.  The  Classification  by  Migula 
BACTERIA   OR   MICROBES 

(Schizomycetes  or  Fission  Fungi) 

i.  Family  COCCACE.E. — Micrococci.     Cells  globular  or  not  elongated. 
Division  in  two  or  three  directions  of  space.     Spore  formation  rare. 

1.  Micrococcus. — Cells  spherical  or  biscuit-shaped.     Division  in  one 
direction  of  space.     With  or  without  flagellae.     A  large  genus,  represented 
by  numerous  species,  pathogenic  and  non-pathogenic,  chromogenic,  zymo- 
genic,  etc. 

2.  Streptococcus. — Generic    limitation    not    clearly    denned.     Often 
merely  chain  forms  of  above,  resulting  from  cohesion  of  cells  dividing  in 
one  direction  of  space. 

3.  Sarcina. — Division  in  three  directions  of  space.     Cells  often  in  fours 
(Tetracoccus) — as  for  example,  the  sarcina  of  the  stomach.     With  or  with- 
out flagellae. 

II.  Family  BACTERIACE.E. — Bacilli.     Cells   more  or  less   elongated, 
cylindrical,  straight;  some  are  somewhat  curved  or  irregular  in  outline. 
With  or  without  flagellae.     Endospore  formation.     Transverse  septation. 

1.  Bacillus. — Variable  in  size  and  length  of  cell.     Numerous  flagellae. 
Endospore  formation  common.     A  very  large  group,  to  which  belong  many 
of  the  most  important  microbes.     Includes  the  old  genus  Bacterium. 

2.  Pseudomonas. — Said  to  have  only  polar  flagellae.     Doubtful  genus, 
by  many  relegated  to  the  group  bacillus. 

III.  Family   SPIRILLACE.E. — Spirillae.     Cells   elongated   and   spirally 
twisted.     Transverse  septation.     Body  fixed,  with  polar  flagellae. 

1.  Spirillum. — Numerous  polar  flagellae.    Large  group. 

2.  Microspira. — Few  polar  flagellae.     A  group  Spirosoma  is  said  to  be 
without  flagellae. 

IV.  Family  SPIROCHETACE.E. — Spirocheta.    Long,  single-celled,  flex- 
ible, spirally  twisted  threads  without  flagellae.     One  genus — Spirocheta. 
(Some  authorities  place  these  organisms  in  the  animal  kingdom  with  the 
Protozoa.) 

V.  Family    MYCOBACTERIACE^E. — Filamentous    organisms,    perhaps 
forming  a  connecting  link  between  bacteria  proper  and  the  lower  filamen- 
tous fungi.     Cells  filamentous  but  not  enclosed  in  a  sheath.     To  this 
family  belong  the  groups  Mycobacterium  and  Actinomyces  (ray  fungus). 
No  flagellae  have  been  observed.     Mostly  transverse  septation.     Gonidial 
(spore)  formation  has  been  observed. 

VI.  Family  CHLAMYDOBACTERIACE.E. — Resembling  above  family,  but 
the  cell  filaments  are  enclosed  in  a  sheath.     The  following  not  very  clearly 


GENERAL  MORPHOLOGY   AND   PHYSIOLOGY  39 

defined  groups  are  recognized:  Cladothrix,  Crenothrix,  Phragmidiothrix, 
and  Thiothrix. 

VII.  Family  BEGGIATOACE^E. — Beggiatoa.  Family  characters  not 
clearly  defined.  Motile,  though  no  flagellae  have  been  observed.  Beg- 
giatoa is  the  most  important  genus. 

Recently  (1917)  the  Society  of  American  Bacteriologists  appointed 
a  committee  on  bacterial  nomenclature  with  instructions  to  look  into  the 
matter  of  bacterial  classification  and  to  propose  a  system  which  would  in 
a  way  represent  the  biological  and  phylogenetic  relationships  of  the  prin- 
ciple groups.  The  following  classification  is  offered  by  this  committee, 
hoping  that  it  may  serve  as  the  basis  for  further  efforts  along  this  line. 

2.  Suggested  Outline  of  Bacterial  Classification 
THE  CLASS  SCHIZOMYCETES 

Minute,  one-celled,  chlorophyll-free,  colorless,  rarely  violet-red  or 
green-colored  plants,  which  typically  multiply  by  dividing  in  one,  two  or 
three  directions  of  space,  the  cells  thus  formed  sometimes  remaining  united 
into  filamentous,  flat,  or  cubical  aggregates.  Filamentous  species  often 
surrounded  by  a  common  sheath.  Capsule  or  sheath  composed  in  the 
main  of  protein  matter.  The  cell  plasma  generally  homogeneous  without 
a  nucleus.  Sexual  reproduction  absent.  In  many  species  resting  bodies 
are  produced,  either  endospores  or  gonidia.  Cells  may  be  motile  by  means 
of  flagella. 

A.  ORDER  MyxoBACTERiALES.1 — Cells  united  during  the  vegetative 
stage  into  a  pseudoplasmodium  which  passes  over  into  a  highly-developed 
cyst-producing  resting  stage. 

B.  ORDER    TmoBACTERiALES.1 — Cells    free    or  united  in  elongated 
filaments.     Water  forms,   not   easily   cultivable.    'Life   energy   derived 
mainly  from  oxidative  processes.     Cells  typically  containing  either  gran- 
ules of  free  sulphur  or  bacterio-purpurin,  or  both,  usually  growing  best 
in  the  presence  of  hydrogen  sulphide. 

C.  ORDER  CnLAMYDOBACTERiALES.1 — Cells  normally  united  in  elon- 
gated filaments.     Sulphur  and  bacterio-purpurin  are  absent.     Iron  often 
present  and  usually  a  well-marked  sheath. 

D.  ORDER    EUBACTERIALES. — Ordo     nov.     Synonyms:       Bacterina 
Perty  1^52  in  part;  Eubacteria  Schroeter  1886;  Eubacteriaceae   A.    J. 
Smith  1902. 

The  order  Eubacteriales  includes  the  forms  usually  termed  the  true 
bacteria,  that  is,  those  forms  which  are  considered  least  differentiated 
and  least  specialized.  The  cell  metabolism  is  not  primarily  bound  up  with 

1  These  first  three  orders  are  included  briefly  to  give  the  complete  setting  of  the 
fourth,  the  Eubaeteriales,  with  which  we  are  primarily  concerned. 


40  PHARMACEUTICAL  BACTERIOLOGY 

hydrogen  sulphide  or  other  sulfur  compounds,  the  cells  in  consequence 
containing  neither  sulfur  granules  nor  bacterio-purpurin.  The  cells 
apparently  do  not  possess  a  well-organized  or  well-differentiated  nucleus. 
The  cells  are  usually  minute  and  spherical,  rod-shaped  or  spiral  in  shape, 
in  most  genera  not  producing  true  filaments;  the  filaments  when  farmed 
not  sheathed,  and  frequently  branching,  thus  being  differentiated  from 
the  iron  bacteria.  The  cells  may  occur  singly,  in  chains  or  other  groupings. 
The  cells  may  be  motile  by  means  of  flagella,  or  non-motile;  they  are  never 
notably  flexuous.  Cell  multiplication  occurs  always  by  transverse,  never 
by  longitudinal  fission.  Some  genera  produce  endospores,  particularly 
the  rod-shaped  types.  More  or  less  branching  of  cells  and  .filaments  may 
occur,  reaching  its  maximum  expression  in  the  genera  Nocardia  and  Acti- 
nomyces  which  may  show  typical  mycelium  formation,  intergrading 
with  the  molds.  Chlorophyll  is  absent,  though  the  cells  may  be  pig- 
mented.  The  cells  may  be  united  into  gelatinous  masses,  but  never  form 
motile  pseudoplasmodia  nor  develop  a  highly  specialized  cyst-producing 
fruiting  stage,  such  as  is  characteristic  of  the  Myxobacteriales. 

I.  Family  NITROBACTERIACE^E. —  Fam.  nov.  Organisms  usually 
rod-shaped  (sometimes  spherical  in  Nitrosomonas  and  possibly  in  Azoto- 
bacter).  Cells  motile  or  non-motile;  when  motile  with  polar,  never  peri  - 
trichous,  flagella.  Endospores  never  formed.  Obligate  aerobes,  capable 
of  securing  growth  energy  by  the  direct  oxidation  of  carbon,  hydrogen 
or  nitrogen  or  of  simply  compounds  of  these.  Non-parasitic  (usually 
water  or  earth  forms). 

1.  Hydrogenomonas.     Jensen,  1909. — Monotrichic  short  rods  capable 
of  growing  in  the  absence  of  organic  matter,  and  securing  growth  energy 
by  the  oxidation  of  hydrogen  (forming  water).     Kaserer  (1905)  who  first 
described  the  organism  states  that  his  species  will  also  grow  well  on  a  variety 
of  organic  substances. 

The  type  species  is  Hydrogenomonas  pqntotropha  (Kaserer)  Jensen. 
Nikleuski  (1910)  described  two  additional  species,  H.  mtrea  and  H.flava. 

2.  Methanomonas.     Jensen,  1909. — Monotrichic  short  rods  capable  of 
growing  in  the  absence  of  organic  matter  and  securing  growth  energy 
by  the  oxidation  of  methane  (forming  carbon  dioxide  and  hydrogen). 
The  type  species  is  Methanomonas  methanica  (Sohngen)  Jensen. 

3.  Carboxydomonas.     Jensen,    1909. — Autotrophic    rod-shaped    cells 
capable  of  securing  growth  energy  by  the  oxidation  of  carbon  monoxide 
(forming  carbon  dioxide).     The  type  species,  Carboxydomonas  oligocar- 
bophila  (Beijerinck  and  van  Delden)  Jensen,  is  described  as  non-motile. 

4.  Mycoderma.     Persoon,  1822  emended. — Synonyms:  UlvinaKuetz- 
ing  1837;  Umbina  Naegeli  1849;  Bacteriopsis?     Trevisan  1885;  Gliacoc- 
cus:  Maggi  1886;  Acetobacter  Eurhmann  1905;  Acetimonas  Jensen  1909. 


GENERAL   MORPHOLOGY   AND   PHYSIOLOGY  41 

Cells  rod-shaped,  frequently  in  chains,  non-motile.  Cells  grow 
usually  on  the  surface  of  alcoholic  solutions,  securing  growth  energy  by 
the  oxidation  of  alcohol  to  acetic  acid.  Also  capable  of  utilizing  certain 
other  carbonaceous  compounds,  as  sugar  and  acetic  acid.  Elongated, 
filamentous,  club-shaped,  swollen  and  even  branched  cells  common  and 
quite  characteristic. 

The  type  species  is  Mycoderma  aceti  Thompson? 

5.  Nitrosomonas.     Winogradsky,  1892. — Includes  Nitrosococcus  Win- 
ogradsky  1892. 

Cells  rod-shaped,  or  spherical,  motile  or  non-motile,  if  motile  with 
polar  flagella.  Capable  of  securing  growth  energy  by  the  oxidation  of 
ammonia  to  nitrates.  Growth  on  media  containing  organic  substances 
scanty  or  absent. 

The  type  species  is  Nitrosomonas  europcea  Winogradsky. 

6.  Nitrobacter.     Winogradsky?     1892. — Synonym:  Nitrosobacterium? 
Rullmann  1897. 

Cells  rod-shaped,  non-motile,  not  growing  readily  on  organic  media 
or  in  the  presence  of  ammonia.  Cells  capable  of  securing  growth  energy 
by  the  oxidation  of  nitrites  to  nitrates. 

Winogradsky  names  no  species,  although  he  described  one.  It  might 
be  termed  Nitrobacter  Winogradskyi  and  made  the  type  species. 

7.  Azotobacter,    Beijerinck,  1901. — Synonyms:  Par achromatium  Beij e- 
rinck  1903;  Azotomonas  Jensen  1909. 

Relatively  large  rods,  or  even  cocci,  sometimes  almost  yeast-like  in 
appearance,  dependent  primarily  for  growth  energy  upon  the  oxidation 
of  carbohydrates.  Motile  or  non-motile;  when  motile,  with  tuft  of  polar 
flagella.  Obligate  aerobes  usually  growing  in  a  film  upon  the  surface  of 
the  culture  medium.  Capable  of  fixing  atmospheric  nitrogen  when  grown 
in  solutions  containing  carbohydrates  and  deficient  in  combined  nitrogen. 
The  best-known  free-living  nitrogen-fixing  bacteria  of  the  soil. 

The  type  species  is  Azotobacter  chroococcum  Beijerinck. 

8.  Rhizobium.    Frank,  1889. — Synonyms :  Phytomyxa  Schroeter  1886; 
Cladochytrium  Vuillemin   1888;  Rhizobacterium  Kirchner   1895;  Pseudo- 
rhizobium  Hartleb   1900;  Rhizomonas  Jensen   1909.     (See  also  p.  104.) 

Comment.  Phytomyxa  Schroeter  has  priority  over  Rhizobium,  but 
because  of  the  confusion  which  would  arise  from  the  substitution  of  the 
older  correct  name  for  the  better  known  term  Rhizobium,  the  committee 
recommends  the  adoption  of  the  latter. 

Minute  rods,  motile  when  young  by  means  of  polar  flagella.  Involu- 
tion forms  abundant  and  characteristic  when  grown  under  suitable 
conditions.  Obligate  aerobes,  capable  of  fixing  atmospheric  nitrogen 
when  grown  in  the  presence  of  carbohydrates  in  the  absence  of  com- 


42  PHARMACEUTICAL  BACTERIOLOGY 

pounds  of  nitrogen.  Produce  nodules  upon  the  roots  of  leguminous 
plants,1 

The  type  species  is  Rhizobium  leguminosarum  Frank. 

II.  Family  MYCOBACTERIACE.E  Chester,  1897. — Cells  usually  elon- 
gated, frequently  filamentous  and  with  a  decided  tendency  to  the  develop- 
ment of  branches,  in  some  genera  giving  rise  to  the  formation  of  a  definite 
branched  mycelium.  Cells  frequently  show  swellings,  clubbed  or  irregu- 
lar shapes.  Endospores  not  produced,  but  conidia  developed  in  some 
genera.  Usually  Gram-positive.  Non-motile.  Many  species  are  para- 
sitic in  animals  or  plants.  Complex  proteins  usually  required.  As  a 
rule  strongly  aerobic  (except  for  some  species  of  Actinomyces  and  the 
genera  Fusiformis  and  Leptotrichia) ,  and  oxidative.  Growth  on  culture 
media  often  slow;  some  genera  show  mold-like  colonies. 

1.  Actinomyces.     Harz,    1877. — Synonyms:  Streptothrix    Cohn   1875, 
not  Streptothrix  Cor  da  1839;  Discomyces  Rivolta  and  Micellone  1878; 
Micromyces  Gruber  1891,  not  Micromyces  Dangeard  1888;  Ob'spora  Sauva- 
geau  and  Radais   1892;  not  Ob'spora  Wallroth   1833;  Cohnistreptothrix 
Pinoy  1913. 

Organism  growing  in  form  of  a  much-branched  mycelium,  which  may 
break  up  into  segments  that  function  as  conidia.  Usually  parasitic,  with 
clubbed  ends  of  radiating  threads  conspicuous  in  lesions  in  animal  body. 
No  earial  hyphae  or  conidia.  Some  species  are  microaerophilic  or  anae- 
robic. Non-motile. 

The  type  species  is  Actinomyces  boms  Harz. 

2.  Nocardia.     Trevisan,    1889. — Synonyms:  Actinomyces    of    many 
authors;    Streptothrix   of  many  authors;    Thermoactinomyces  Tsilinsky 
1899. 

Branched  filaments,  resembling  a  mycelium,  readily  breaking  up  into 
segments,  usually  saprophytic  soil  forms.  Differs  primarily  from  Acti- 
nomyces in  the  development  of  aerial  hyphae  and  conidia.  Usually 
aerobic.  Many  are  pigment  formers.  Colonies  as  a  rule  mold-like  on 
culture  media. 

3.  Mycobacterium.    Lehmann     and     Neumann,     1896. — Synonyms: 
Sclerothrix  Metschnikoff  1888,  not  Sclerothrix  Kuetzing  1849;  Coccothrix 
Lutz  1886;  Mycomonas  Jensen  1909. 

Slender  rods  which  are  stained  with  difficulty,  but  when  once  stained 
are  acid-fast.  Cells  sometimes  show  swollen,  clavate  or  cuneate  forms, 
and  occasionally  even  branched  filaments.  Non-motile,  Gram-positive. 
No  endospores.  Growth  on  media  slow.  Aerobic.  Several  species 
pathogenic  to  animals. 

1True  nodule  forming  bacteria  occur  in  the  shrub  Ceanothus  integerrimus  and 
probably  ether  non-leguminous  plants. — Author. 


GENERAL   MORPHOLOGY   AND   PHYSIOLOGY  43 

The  type  species  is  Mycobacterium  tuberculosis  (Koch)  Lehmann  and 
Neumann. 

4.  Corynebacterium.    Lehmann    and    Neumann,     1896. — Synonyms: 
Corynemonas  Jensen  1909;  Corynethrix  Bingert  1901. 

Slender,  often  slightly  curved,  rods  with  tendency  to  club  formation ,- 
branching  cells  occasionally  seen  in  old  cultures.  Barred  irregular  stain- 
ing. Not  acid-fast.  Gram-positive.  Non-motile.  Aerobic.  No  en- 
dospores.  Some  pathogenic  species  produce  a  powerful  exotoxin. 
Characteristic  snapping  motion  is  exhibited  when  cells  divide. 

The  type  species  is  Corynebacterium  diphtheria  (LoefHer)  Lehmann  and 
Neumann. 

5.  Fusiformis.     Hoelling,  1910. — Synonym:  Mantegazzaea  Vuillemin 
1913,  not  Mantegazzaea  Trevisan  1879. 

Obligate  parasites.  Cells  usually  elongate  and  fusiform,  staining 
somewhat  irregularly.  Filaments  sometimes  formed;  non-branching. 
Non-motile.  No  spores.  Growth  in  laboratory  media  feeble. 

The  type  species  (?)  is  Fusiformis  termitidis  Hoelling. 

6.  Leptotrichia.     Trevisan,    1879    emended. — Synonyms:  Leptothrix 
Robin  1847,  not  Leptothrix  Kuetzing  1843;  Rasmussenia  Trevisan  1889. 

Thick,  long  straight  or  curved  threads,  frequently  clubbed  at  one  end 
and  tapering  to  the  other.  Gram-positive  when  young.  Threads 
fragment  into  short,  thick  rods.  Anaerobic  or  facultative.  Non-motile. 
Filaments  sometimes  granular;  non-branching.  No  aerial  hyphae  or 
conidia.  Parasites  or  facultative  parasites. 

The  type  species  is  Leptotrichia  buccalis  (Robin)  Trevisan. 

III.  Family  PSEUDOMONADACE.E. — Short  rods,  usually  motile.     Flag- 
ella  single,  polar.     Gram-negative.     Not  obligate  aerobes.     Many  species 
active  ammonifiers.     Many  species  produce  water-soluble  pigments  or 
green  fluorescence;  yellow  pigment  common.     Some  species  are  photo- 
genic.    Soil  and  water  bacteria,  with  many  plant  parasites. 

i.  Pseudomonas.  Migula,  1894. — Synonyms:  Bactrillum  Fischer  1895; 
Arthrobactrillum  Fischer  1895;  Eupseudomonas  Migula  1895;  Bactrinius 
Kendall  1902;  Bactrillius  Kendall  1902;  Bacterium  Ehrenberg  emended 
E.  F.  Smith  1905;  Denitromonas  Jensen  1909; Liquidomonas  Jensen  1909. 
'  Rod-shaped,  short,  usually  motile  by  means  of  polar  flagella  or  rarely 
non-motile.  Aerobic  and  facultative.  Frequently  gelatin  liquefiers  and 
active  ammonifiers.  No  endospores.  Gram  stain  variable,  though  usu- 
ally negative.  Fermentation  of  carbohydrates  as  a  rule  not  active.  Fre- 
quently producing  a  water-soluble  pigment  which  diffuses  through  the 
medium  as  green,  blue,  purple,  brown,  etc.  In  some  cases  a  non-diffusible 
yellow  pigment  is  formed.  Many  yellow  species  are  plant  parasites. 

IV.  Family    SPIRILLACE^E  Migula,  1894. — Cells   elongate,    more  or 


44  PHARMACEUTICAL  BACTERIOLOGY 

less  spirally  curved.  Cell  division  always  transverse,  never  longitudinal. 
Cells  non-flexuous.  Usually  without  endospores.  As  a  rule  motile  by 
means  of  polar  flagella,  sometimes  non-motile.  Typically  water  forms, 
though  some  species  are  intestinal  parasites. 

1.  Vibrio  Miller,  1773  emended  E.  F.  Smith,  1905. — Synonyms:  Pacinia 
Trevisan  1885;  M icrospira Schroeter  1886;  PseudospiraDeTomandTiGvi- 
san   1889;  Liquidovibrio  Jensen   1909;  Solidombrio  Jensen   1909;  Photo- 
bacterium?     Beijerinck  1889. 

Cells  short  bent  rods,  rigid,  single  or  united  into  spirals.  Motile 
by  means  of  a  single  (rarely  two  or  three)  polar  flagellum,  which  is  usually 
relatively  short.  Many  species  liquefy  gelatin  and  are  active  ammonifiers. 
Aerobic  and  facultative.  No  endospores.  Usually  Gram-negative. 
Water  forms,  a  few  parasites. 

The  type  species  is  Vibrio  cholera  (Koch)  Buchner. 

2.  Spirillum.     Ehrenberg,  1930  emended  Migula,  1894. — Synonyms: 
Spirobacillus?     Metschnikoff  1889;  Spirosoma  Migula  1894;  Sporospiril- 
lum?    Jensen  1909. 

Cells  rigid  rods  of  various  thicknesses,  length,  and  pitch  of  the  spiral, 
forming  either  long  screws  or  portions  of  a  turn.  Cells  motile  by  means  of 
a  tuft  of  polar  flagella  (5  to  20)  which  are  mostly  half  circular,  rarely  wavy- 
bent.  These  flagella  occur  on  one  or  both  poles;  their  number  varies 
greatly  and  is  difficult  to  determine,  since  in  stained  preparations  several 
are  often  united  into  a  common  strand.  Endospore  formation  has  been 
reported  in  some  species.  Habitat;  water  or  putrid  infusions. 

V.  Family  COCCACE.E  Zopf,  1884  emended  Migula,  1894. — Synonyms; 
Sphaerobacteria  Cohn  1872;  Coccogena  Trevisan  1885;  Coccacei  Schroeter 
1886;  Coccobacteria  Schroeter  1886. 

Cells  in  their  free  conditions,  spherical;  during  division  somewhat 
elliptical.  Division  in  one,  two  or  three  planes.  If  the  cells  remain  in 
contact  after  division  they  are  frequently  flattened  in  the  plane  of  divi- 
sicn.  Motility  rare.  Endospores  absent.  Metabolism  complex,  usually 
involving  the  utilization  of  amino-acids  or  carbohydrates. 

Tribe  i.  STREPTOCOCCE.E  Trevisan. — Parasites  (thriving  only  or 
best  on  or  in  the  animal  body).  Grow  well  under  anaerobic  conditions. 
Many  forms  grow  with  difficulty  on  media,  none  very  abundantly.  Planes 
of  fission  usually  parallel,  producing  pairs  or  short  or  long  chains,  never 
packets.  Generally  stain  by  Gram.  Produce  acid  but  no  gas  in  glucose 
and  lactose  broth.  Pigment,  if  any,  white  or  orange. 

i.  Neisseria.  Trevisan,  1885. — Synonyms:  Diplococcus  Weichsel- 
baum  1886  in  part;  Gonococcus?  Neisser  ?  1879;  Merismopedia  Zopf 
1885,  not  Merismopedia  Meyen  1839. 

Strict  parasites,  failing  to  grow  or  growing  very  poorly  on  artificial 


GENERAL   MORPHOLOGY  AND   PHYSIOLOGY  45 

media.  Cells  normally  in  pairs  of  flattened  cells.  Gram-negative. 
Fermentative  powers  low.  Growth  fairly  abundant  on  serum  media, 
usually  whitish  or  yellowish. 

The  type  species  is  Neisseria  gonorrhoea  Trevisan. 

2.  Streptococcus.     Rosenbach,    1884,  emended  Winslow  and  Rogers, 
1905. — Synonyms:   Sph&rococcus    Marpmann    1885,    not    Sph&rococcus 
Agardh    1821;   Perroncitoa   Trevisan    1889;    Babesia?    Trevisan    1889; 
Schuetzia  Trevisan  1889;  Lactococcus  Beijerinck  1901;  Hypnococcus  Bet- 
tencourt  et  al.     1904;  Myxokokkus  Gonnermann  1907,  not  Myxococcus 
Thaxter  1892;  Melococcus?     Amiradzibi  1907;  Diplostreptococcus  Lingels- 
heim  1912. 

Chiefly  parasites.  Cells  normally  in  short  or  long  chains  (under  un- 
favorable conditions,  sometimes  in  pairs  and  small  groups,  never  in  large 
packets).  Generally  stain  by  Gram.  Capsules  and  zooglea  often  formed. 
On  agar  streak,  effused  translucent  growth,  often  with  isolated  colonies. 
In  stab  culture,  little  surface  growth.  Sugars  fermented  with  formation 
of  large  amount  of  acid.  Generally  fail  to  liquefy  gelatin  or  reduce  nitrates. 

Type  species  is  Streptococcus  pyo genes  Rosenbach. 

3.  Staphylococcus.     Rosenbach,  1884. — Synonyms:  Micrococcus  Cohn 
1872  em.  Migula  1894;  Botryomyces  Bollinger  1888;  Botryococcus  Kitt  1888, 
not  Botryococcus  Kuetzing   1849;   Galactococcus   Guillebeau;    Bollingera 
Trevisan  1889;  Gaffkya  Trevisan  1885;  Pyococcus  Ludwig  1892;  Carpho- 
coccus  Hohl  1902;  Aurococcus  Winslow  and  Rogers  1906,  Indolococcus 
Jensen   1909;  Liquidococcus  Jensen   1909;  Peptonococcus  Jensen   1909; 
Enterococcus?     (Thiercelin)  Rougentzoff  1914. 

Parasites.  Cells  in  groups  and  short  chains,  very  rarely  in  packets. 
Generally  stain  by  Gram.  On  agar  streak  good  growth,  of  orange  color. 
Sugars  fermented  with  formation  of  moderate  amount  of  acid.  Gelatin 
often  liquefied  very  actively. 

Type  species  is  Staphylococcus  aureus  Rosenbach. 

4.  Albococcus.     Winslow  and  Rogers,  1905. — Differs  from  Staphylo- 
coccus in  forming  more  abundant  surface  growth  of  porcelain  white  color, 
and  in  fact  that  liquefaction  of  gelatin  when  present  is  less  vigorous. 

Tribe  2.  Micrococcea.  Trevisan. — Facultative  parasites  or  sapro- 
phytes. Thrive  best  under  aerobic  conditions.  Grow  well  on  artificial 
media,  producing  abundant  surface  growths.  Planes  of  fission  often  at 
right  angles;  cell  aggregates  in  groups,  packets  or  zooglea  masses.  Gen- 
erally decolorize  by  Gram.  Pigment  yellow  or  red. 

5.  Micrococcus.    Cohn,  1872,  emended  Winslow  and  Rogers,  1905. — 
Synonyms:   Micros  phara  Cohn   1872,   not  Microsph&ra  Leveille   1851; 
Pediococcus  Balcke  1884;  Merista  Van  Tieghem  1884,  not  Merista  (Banks 
and  Soland)   Cunningham   1839;  Planococcus  Migula   1894;   Urococcus 


46  PHARMACEUTICAL  BACTERIOLOGY 

Miquel  1879,  not  Urococcus  Kuetzing  1849;  Pedioplana  Wolff  1907; 
Tetradiplococcus?  Bartoszewicz  and  Schwarzwasser  1908;  Solidococcus 
Jensen  1909;  Planomerista  Vuillemin  1913. 

Facultative  parasites  or  saprophytes.  Cells  in  plates  or  irregular 
masses  (never  in  long  chains  or  packets).  Generally  decolorize  by  Gram. 
Growth  on  agar  abundant,  with  formation  of  yellow  pigment.  Glucose 
broth  slightly  acid,  lactose  broth  generally  neutral.  Gelatin  frequently 
liquefied,  but  not  rapidly. 

The  type  species  is  Micrococcus  luteus  (Schroeter)  Cohn. 

6.  Sarcina.     Goodsir,  1842,  emended  Winslow  and  Rogers,  1905. — 
Synonyms:  Urosarcina  Miquel   1879;  Planosarcina  Migula   1849;  Lac- 
tosarcina  Beijerinck  1908;  Sporosarcina?    Jensen  1909. 

i  Sarcina  differs  from  Micrococcus  solely  in  fact  that  cell  division 
occurs  under  avorable  conditions  in  three  planes,  forming  regular 
packets. 

The  type  species  is  Sarcina  ventriculi  Goodsir. 

7.  Rhodococcus.     Fliigge,  1891,  emended  Winslow  and  Rogers,  1906. — 
Synonyms:  Not  Rhodococcus  Molisch  1907. 

Saprophytes.  Cells  in  groups  or  regaar  packets.  Generally  de- 
colorize by  Gram.  Growth  on  agar  abundant  with  formation  of  red 
pigment.  Glucose  broth  lightly  acid,  lactose  broth  neutral.  Gelatin 
rarely  liquefied.  Nitrates  generally  reduced  to  nitrites. 

VI  Family  BACTERIACE^  Cohn,  1872,  emended. — Rod-shaped 
cells  without  endospores.  Gram-negative.  Flagella  when  present  peri- 
trichic.  Metabolism  complex,  amino-acids  being  utilized,  and  generally 
carbohydrates. 

1.  Bacterium.     Ehrenberg,  1838,  emended  Jensen,  1909. — Synonyms: 
Actinobacter  Duclaux  1882  in  part;  Klebsiella  Trevisan   1885  in  part; 
G  iscrobacterium  Malerba  and  Sanna  Salaris  1888;  Aerobacter  Beijerinck 
1900;  Salmonella  Lignieres  1900;  Denitrobacterium  Jensen  1909. 

Notik  or  non-motile  rods,  staining  evenly.  Easily  cultivable.  Ani- 
mal pathogens  or  saprophytes.  Often  chromogenic.  Many  forms  ac- 
tively decompose  carbohydrates. 

The  type  species  is  Bacterium  coli  Escherich. 

2.  Erwinia.     Nov.  gen. — Plant  pathogens,  Growth  usually  whitish, 
often  slimy.     Indol  generally  not  produced.     Acid  usually  formed  in 
certain  carbohydrate  media,  but  as  a  rule  no  gas. 

3.  Pasteur  el  a.     Trevisan,    1887. — Synonyms:    Octopsis?     Treviean, 
1885;  Coccobacillus  Gamaleia  1888,  not  CoccobacillusLeube  1885. 

Short  rods,  single  or  rarely  in  chains,  usually  showing  distinct  polar 
staining.  Non-motile.  Gram-negative.  Without  spores.  Aerobic  and 
facultative.  Powers  of  carbohydrate  fermentation  slight;  no  gas  produced. 


GENERAL   MORPHOLOGY  AND   PHYSIOLOGY  47 

Gelatin  not  liquefied.  Parasitic,  frequently  pathogenic,  producing  plague 
in  man  and  hemorrhagic  septicemia  in  the  lower  animals. 

The  type  species  is  Pasteur ella  cholera-gallinarum  (Fliigge)  Trevisan. 

4.  Hemophilus.  Gen.  no v. — Synonyms :  Pyobacillus  ?  Koppanyi  1907; 
Diplobacillus  Morax  1896,  not  Diplobacillus  Weichselbaum  1887. 

Minute  rod-shaped  cells,  non-motile,  without  spores,  strict  parasites, 
growing  best  (or  only)  in  the  presence  of  hemoglobin,  and  in  general  re- 
quiring blood  serum  or  ascitic  fluid.  Gram-negative. 

The  type  species  is  Hemophilus  Influenza  (Pfeiffer). 

VII.  Family  LACTOBACILLACE^:.     Fam.  nov. — Rods,  often  long  and 
slender,  Gram-positive,  non-motile,  without  endospores.     Usually  pro- 
duce acid  from  carbohydrates,  as  a  rule  lactic.     When  gas  is  formed,  it 
is  CO2  without  H2.     The  organisms  are  usually  somewhat  thermophilic. 
As  a  rule  microaerophilic;  surface  growth  on  media  poor. 

i.  Lactobacillus.  Beijerinck,  1901.  —  Synonyms:  Dispora?  Kern 
1882;  Saccharobacillus?  van  Laer  1889;  Streptobacillus  Rest  and 
Khoury  1902;  Brachybacterium  Trioli-Petersson  1903;  Caseobacterium 
Jensen  1909. 

Generic  characters  those  of  the  family. 

The   type   species   is  Lactobacillus   caucasicus    (Kern?)     Beijerinck. 

VIII.  Family  BACITLACE^E. — Rods    producing    endospores,    usua  ly 
Gram-positive.     Flagella  when  present  peritrichic.     Actively  decompose 
prote'n  media  through  the  agency  of  enzymes. 

1.  Bacillus.    Cohn,i872. — Synonyms:  Bactrella?    M.orrtmS^o')Metal- 
lacter?     Perty  1852;  Bactridium  Davaine  1868  in  part;  Urobacillus  Miquel 
1879;  Pollendera  Trevisan  1884;  Zopfiella  Trevisan  1885;  Streptobacter 
Schroeter  1886;  Cornilia  Trevisan  1889;  in  part;  Bacterium  Ehrenberg, 
emended  Migula  1894  in  part;  Bactridium  Fischer  1895,  not  Bactridium 
Wallroth    1832;    Bactrinium   Fischer    1895;    Bactrillum    Fischer    1895; 
Endobacterium   Lehmann    and   Neumann    1896;    Astasia  Meyer    1898; 
Fenobacter  Beijerinck  1900;   Bacterius  Kendall  1902  in  part;   Aplano- 
bacter  E.  F.  Smith  1905  in  part;  Semiclostridium  Maassen  1905;  Pienno- 
bakterium  Gonnermann  1907;  Myxobacillus  Gonnermann  1907;  Thermo- 
bacillus   Jensen    1909;   Serratia  Vuillemin  1913   in  part,   not   Serratia 
Bizio  1823. 

Aerobic  forms.  Mostly  saprophytes.  Liquefy  gelatin.  Often  occut 
in  long  threads  and  form  rhizoid  colonies.  Form  of  rod  usually  nor 
greatly  changed  at  sporulation. 

The  type  species  is  Bacillus  subtilis  Cohn. 

2.  Clostridium.     Prazmowski,  1880. — Synonyms;  Amylobacter  Trecul 
1865;  Cornilia  Trevisan   1889  in  part;  Granulobacter  Beijerinck   1893; 
Clostnlum  Fischer  1895;  Clostrinium  Fischer  1895;  Paracloster  Fischer 


48  PHARMACEUTICAL  BACTERIOLOGY 

1895;  Semiclostridium  Maassen  1905;  Botulobacillus  Jensen  1909;  Butryi- 
bacillus  Jensen  1909;  Cellulobacillus  Jensen  1909;  Putribacillus  Jensen 
1909. 

Anaerobes.  Often  parasitic.  Rods  frequently  enlarged  at  sporulation, 
producing  clostridium  or  plectridium  forms. 

The  type  species  is  Clostridium  butyricum  Prazmowski. 

ORGANISMS  INTERMEDIATE  BETWEEN  BACTERIA  AND  PROTOZOA 

Spiroch&tacece.  Swellengrebel,  1907. — Free  living  or  parasitic  spirilli- 
form  organisms  with  or  without  flagella,  with  undulating  or  rigid  spiral 
twists.  Reproduction  by  transverse  division  and  by  "coccoid  bodies," 
the  equivalent  of  spores. 

Four  genera  are  recognized  as  follows : 

1.  Spirochata.     Ehrenberg. — Non-parasitic,  with  flexible  undulating 
body  and  with  or  without  flagelliform  tapering  ends.     Common  in  sewage 
and  foul  waters. 

The  type  species  is  Spirochata  plicatilis  Ehrenberg. 

2.  Cristispira.     Gross. — Giant  forms  with  undulating  body  and  pecul- 
iar flattened  ridge  erroneously  called  an  " undulating  membrane"  which 
runs  the  length  of  the  body.     Parasitic  in  molluscs. 

The  type  species  is  Cristispira  balbianii  Certes,  from  the  crystalline 
style  of  the  oyster. 

3.  Saprospira.     Gross. — Non-parasitic  forms  similar  to  Cristispira, 
but  without  the  flattened  ridge  or  "crista"  which  is,  if  present,  here 
replaced  by  a  straight  columella  or  thickening  of  the  periplast. 

The  type  species  is  Saprospira  grandis  Gross. 

4.  Treponema.     Schaudinn. — Parasitic    and    frequently    pathogenic 
forms  with  undulating  or  rigid  spirilliform  body.     Without  crista  or 
columella.     With  or  without  flagelliform  tapering  ends. 

The  type  species  is  Treponema  pallidum  Schaudinn. 

i.  ARTIFICIAL  KEY  TO  THE  ORDERS  or  THE  SCHIZOMYCETES 

Cells  united  during  the  vegetative  stage  into  a 

pseudoplasmodium A.  Myxobacteriales 

Cells  not  forming  a  pseudoplasmodium 

Cells  free  or  united  in  elongated  filaments,  often  with  a  well-defined 
sheath.  Conidia  frequently  formed.  Free  sulphur,  iron  or  bacterio- 
purpurin  often  present. 

Cells  typically  containing  granules  of  sulphur  or  bacterio-purpurin  or 

both B.  Thiobacteriales 

Sulphur  and  bacterio-purpurin  absent;  iron  often 
present C.  Chlamydobacteriales 


GENERAL   MORPHOLOGY   AND   PHYSIOLOGY  49 

Cells  never  in  sheathed  filaments.     Conidia  only  in  the  mycelial. 

Mycobacteriaceae.     Flagella    often  present.     Free  iron,  sulphur,    or 

bacterio-purpurin  never  present D.  Eubacteriales 

2.  ARTIFICIAL  KEY  TO  THE  FAMILIES  OF  THE  EUBACTERIALES 

Cells  spiral  with  polar  flagella IV.  Spirillace® 

Not  as  above 

Cells  spherical;  rarely,  if  ever,  motile;  spores  never  produced;  never 
securing  growth  energy  from  nitrogen  or  ammonia  .    .   V.  Coccaceae 
Not  as  above 

Cells  short  rod-shaped  with  a  single  rarely  two  polar  flagellum;  usually 
forming  green  or  yellow  pigment   ....   III.  Pseudomonadaceae 
Not  wholly  as  above 

Spores  formed VIII.  Bacillaceae 

Spores  never  formed 

Metabolism  simple,  securing  growth  energy  form  carbon,  hydrogen 
or  their  simple  compounds ;  flagella,  if  present, 

polar I.  Nitrobacteriaceae 

Metabolism  complex,  dependent  upon  more  complex  carbohydrate 
and  protein  substances;  flagella,  if  present,  peritrichic.  Cells 
clubbed,  fusiform,  filamentous,  branching  or  mycelial;  those  not 
distinctly  so  are  either  acid-fast  or  show  barred  irregular 

staining II.  Mycobacteriaceae 

Not  as  above 

Gram-positive;  non-motile VII.  Lactobacillaceae 

Gram-negative;  often  motile VI.   Bacteriaceae 

3.  ARTIFICIAL  KEY  TO  THE  GENERA  OF  THE  EUBACTERIALES 

I.  Nitrobacteriaceae 

Fixing  nitrogen  or  oxidizing  its  compounds 
Fixing  nitrogen 

Cells  large;  in  soil 7.  Azotobacter 

Rods  minute;  in  roots  of  leguminous  plants   ....    8.  Rhizobium 
Oxidizing  nitrogen  compounds 

Oxidizing  ammonia 5.  Nitrosomonas 

Oxidizing  nitrites  .    .    . 6.  Nitrobacter 

Not  as  above 

Oxidizing  carbon  compounds 

Oxidizing  alcohol;  branching  forms  common  ...  4.  Mycoderma 
Not  as  above,  using  simpler  carbon  compounds 

Oxidizing  CO 3.  Carboxydomonas 

Oxidizing  CH4 2.  Methanomonas 

4 


50  PHARMACEUTICAL  BACTERIOLOGY 

II.  Mycobacteriaceae 

Slender  rods,  staining  with  difficulty  and  acid-fast  3.  Mycobacterium 
Not  as  above 

Mycelium  and  conidia  formed 

With    aerial    hyphae    and    conidia;    usually    saprophytic    soil 

organisms 2.  Nocardia 

Hyphae  and  conidia  not  aerial;  usually  parasitic  in 

animals i.  Actinomyces 

Not  as  above;  cells  rod-like,  usually  somewhat  curved,  clubbed, 
fusiform,  or  even  branched,  but  never  mycelial. 
Thick,  long  threads,  fragmenting  into  short  thick 

rods Leptotrichic 

Not  as  above 

Cells  usually  elongate  and  fusiform;  filaments,  if  formed,  not 
branching;  staining  somewhat  irregularly  .    .5.  Fusiformis 
Cells  slightly  curved,  clubbed,  or  in  old  cultures  even  branching; 
not  filamentous;  showing  definitely  barred 

staining 4.  Corynebacterium 

III.  Pseudomonadaceae 

Generic  characters  mainly  those  of  family  ...     i.  Pseudomonas 

IV.  Spirillaceae 

Flagellum    single    (rarely    2    or    3) i.  Vibrio 

Flagella  tufted  (5-20) 2.  Spirillum 

V.  Coccaceae 

Abundant  re-pigmented  growth  on  agar 7.  Rhodococcus 

Not  as  above 
Gram-negative 
Normally  in  paris  of  flattened  cells;  growth  on  plain  agar  scanty, 

never  bright  yellow i .  Neisseria 

Normally  in  plates,  packets,  or  irregular  masses,  growth  on  plain 
agar  abundant,  pigment  definitely  yellow. 

Cells  in  regular  packets 6.  Sarcina 

Cells  not  in  regular  packets 5.  Micrococcus 

Gram-positive   (Exceptions    rare  and  not  easily  confused  with 
above  genera). 

Cells  normally  in  chains,  sometimes  in  paris  (especially  in  acid 
environment)  never  in  large  irregular  masses.     Gelatine  rarely 
liquefied. 
Growth  on  plain  agar  usually  translucent,  never  heavy,  never 

yellow  or  orange 2.  Streptococcus 

Cells   normally  in  groups  or  masses   (occasionally  in  plates  in 


GENERAL   MORPHOLOGY  AND   PHYSIOLOGY  ,  51 

Albococcus?);  chains  short  and  irregular,  if  present.     Gelatine 

often  liquefied. 

Agar  growth  abundant,  white  to  orange 

Pigment    orange    (rarely   lacking)    gelatine    often   liquefied 

actively 3.  Staphylococcus 

Whitish  to  porcelain  white;  liquefaction  less 

vigorous 4.  Albococcus 

VI.  Bacteriaceae 

Plant   pathogens  .v 2.  Erwinia 

Not  as  above;  saprophytes  or  in  animal  habitats  (intestines,  tissues, 

etc.). 
Usually  motile  and  exhibiting  active  fermentative  powers;  typically 

parasitic  in  intestines  of  man  and  higher  animals;  growing  well 

on   ordinary  media i.  Bacterium 

Not  wholly  as  above 

Growing  only  in  presence  of  hemoglobin,  ascitic  fluid  or 

serum 4.  Hemophilus 

Growth  on  media  scanty,  but  less  sensitive  than  the  above ;  short  rods 

with  tendency  to  bipolar  stain 3.  Pasteurella 

VII.  Lactobacillacese 

Generic  characters  mainly  those  of  family  .    .    .   i.  Lactobacillus 

VIII.  Bacillaceae 

Aerobic,  usually  saprophytic,  cells  not  greatly  enlarged  (if  at  all) 
at  sporulation i.  Bacillus 

Anaerobic,  often  sprophytic,  cells  frequently  enlarged  at 

sporulation 2.  Clostridium 

3.  General  Morphology  of  Microbes 

As  already  stated,  the  morphology  of  microbes  is  simple.  They  con- 
sist of  a  single  cell  composed  of  cell- wall  and  cell-contents.  The  cell-wall 
consists  of  cellulose,  and  is  very  thin;  stains  readily  with  the  various 
bacterial  stains.  The  chief  cell-contents  is  the  cytoplasmic  or  protoplas- 
mic living  base  commonly  designated  as  the  nucleoplasm,  which  is  of  a 
granular  nature,  and  by  some  is  supposed  to  be  a  nucleus  in  a  divided  state. 
A  nucleus  proper  does  not  exist,  or,  rather,  has  not  been  demonstrated. 
The  cytoplasm,  as  a  rule,  stains  quite  readily.  Distributed  through  the 
cytoplasm  may  be  found  various  substances,  elaborated  by  cytoplasmic 
activity.  Polar  granules  (metachromes  or  Babes-Ernest  granules)  have 
been  observed.  Sulphur,  fat,  pigment,  chlorophyll,  etc.,  may  be  found. 


PHARMACEUTICAL  BACTERIOLOGY 


The  cell- walls  of  many  species  undergo  a  gelatinous  change.  This 
change  may  affect  the  outer  layers  only,  or  it  may  involve  the  entire  thick- 
ness of  the  wall,  forming  the  gelatinous  substances  noticeable  in  bacterial 
cultures  and  in  other  substances  (stringy  cultures,  stringy  milk,  etc.). 
This  gelatinous  substance  also  causes  the  individual  organisms  to  cling  to 
each  other,  thus  causing  the  formation  of  the  peculiar  zooglea  masses  in 
in  natural  as  well  as  in  artificial  culture  media. 


FIG.  4. — Illustrating  the  general  morphology  of  microbes,  a,  showing  general 
structure  of  a  bacillus,  endospore  formation,  and  development  of  new  bacillus  from  a 
spore;  b,  showing  manner  of  transverse  septation;  c,  arrangement  of  nagellae,  single  uni- 
polar, single  bipolar;  and  multiple,  polar  and  general;  d,  cocci;  e,  nagellae  of  cocci;/, 
spirillum  with  single  polar  cilia. 

The  cilia  or  flagellae  are  very  delicate  threads,  supposed  to  extend  from 
the  cell-plasm,  through  the  cell-wall,  into  the  surrounding  medium.  The 
delicate  threads  are  probably  cytoplasmic  in  nature,  and  by  their  rapid 
vibratory  motion  enable  the  microbe  to  move  about  within  liquid  media. 
Some  microbes  are  apparently  without  flagellae,  nor  is  it  definitely  deter- 
mined that  all  motile  microbes  have  flagellae.  The  attempt  to  make 
generic  distinctions  based  upon  the  absence  or  presence  of  few  or  many 
flagellae,  upon  the  existence  of  polar  or  non-polar  flagellae,  etc.,  is  unsatis- 
factory. Special  staining  methods  are  necessary  to  demonstrate  the 
presence  or  absence  of  flagellae. 


GENERAL   MORPHOLOGY   AND    PHYSIOLOGY 


53 


FIG.  5. — Illustrating  the  general  morphology  of  Coccaceae.  a,  b, '  micrococci  (a) 
differing  in  size,  showing  chain  formation  or  streptococci  (b);  c,  diplococcus;  d,  diplococ- 
cus;  e,  tetracoccus ; /,  gelatinized  tetracoccus;  g,  gelatinized  diplococcus. 


PIG.  6. — General  morphology  of  Bacteriaceae.  a,  b,  c,  d,  bacilli  differing  in  size  and 
form;  c,  shows  curved  bacilli  like  those  of  Asiatic  cholera;  e,  hay  bacillus  (B.  subtilis); 
/,  Y-shaped  or  branched  bacilli,  as  of  clover  root  nodules;  g,  drum-stick  (Trommel- 
achlager)  bacilli,  as  of  tetanus — form  due  to  the  enlarged  endospores. 


54 


PHARMACEUTICAL  BACTERIOLOGY 


The  rate  of  motion  of  bacteria  has  been  measured.     The  cholera  bacil- 
lus moves  at  the  rate  of  18  cm.  per  hour.     The  typhoid  bacillus  is  slower 


FIG.  7. — General  morphology  of  the  Spirillaceae.  a,  S-shaped  or  single  spiral;  b, 
double  spiral;  c,  multiple  spirals;  d,  slender  threads;  a  and  b  have  fixed  bodies,  motion 
being  caused  by  flagellae;  c  and  d,  bodies  flexible,  motion  not  due  to  flagellae. 

moving  a  distance  of  4  mm.  in  one  hour.    The  rate  of  motion  in  one  and 
the  same  species  is,  however,  variable,  being  comparatively  rapid  at  one 


FIG.  8. — Illustrating  polymorphism  or  pleomorphism.     Involution  forms  of  the  bacillus 
of  Asiatic  cholera.     (Williams.) 

time  under  certain  conditions  of  food  supply,  warmth,  etc.,  and  at  other, 
times  comparatively  slow. 


GENERAL   MORPHOLOGY   AND   PHYSIOLOGY  55 

When  the  bacteria  approach  the  end  of  the  life  cycle,  or  when  the 
conditions  for  growth  and  septation  become  unfavorable,  spore  formation 
may  take  place.  However,  not  all  species  of  bacteria  form  spores  (endo- 
spores).  For  example,  most  of  the  pathogens  do  not  form  spores.  Each 
cell  forms  one  spore  only,  there  being  apparently  no  exception  to  this 
rule.  In  most  cases  the  spore  occupies  a  position  nearer  one  end  of  the 
cell,  more  rarely  it  occurs  in  a  median  position.  As  to  form  the  spore  is 
generally  somewhat  oval  in  the  direction  of  the  long  axis  of  the  cell.  It 
may  be  more  or  less  irregular  in  outline,  as  in  Bacillus  botulinus,  the  cause 
of  botulism.  The  cell  which  contains  a  median  spore  causing  a  bulging  of 
the  cell  is  called  a  clostridium.  If  it  causes  a  terminal  bulging  it  is  called 


4 


PIG.  9. — Illustrating  polymorphism  or  pleomorphism.  a  to  d,  inclusive,  represent 
different  forms  of  the  same  organism — the  Diphtheria  bacillus.  (See  also  Figs.  46-50 
inclusive.) 

a  plectridium,  also  drum  stick  bacillus  (Trommelschlager  bacillus).  The 
spore  is  formed  from  the  cell  plasm,  and  differs  from  it  in  its  higher  re- 
fractive index  and  its  peculiar  resistance  to  the  action  of  stains.  As  soon 
as  spore  formation  is  complete,  the  rest  of  the  cytoplasm  dies,  the  cell- 
wall  disintegrates,  and  the  spore  is  thus  set  free.  Spores  have  a  remark- 
able resisting  power  to  high  temperatures  and  other  unfavorable  conditions. 
In  a  dry  atmosphere  they  may  lie  dormant  for  a  long  time,  even  several 
years.  Boiling  from  one  to  two  hours  does  not  kill  some  of  them  (spores 
of  hay  bacillus).  As  soon  as  the  spores  are  placed  in  suitable  media 
(adequate  warmth,  moisture,  and  food  supply)  they  develop  into  new 
individuals,  which  continue  to  septate  until  spore  formation  again  takes 
place. 

The  classification  given  above,  into  families  and  genera,  and  Figs.  2  to 
10,  inclusive,  will  serve  to  give  a  fairly  good  idea  of  the  general  structural 
characteristics  of  microbes. 


CALIFORNIA  COtiEtt 


PHARMACEUTICAL  BACTERIOLOGY 


4.  General  Physiology  of  Microbes 

Microbes,  in  common  with  living  things  generally,  spring  from  pre- 
existing parents,  take  in  and  assimilate  food,  grow  and  multiply,  and  finally 
die.  The  rate  of  growth  and  of  multiplication  (septa tion  or  division) 
varies  somewhat,  depending  on  temperature,  moisture,  and  food  supply. 
The  average  life  of  one  individual  (from  division  to  division)  is  perhaps 
thirty  minutes.  Under  favorable  condition  the  period  is  much  shortened. 
This  life  period  of  the  individual  cell  must  not  be  confounded  with  the  life 
cycle  of  the  individuals  resulting  from  a  single  cell  or  parent.  It  is  known 
that  under  uniform  conditions  of  temperature,  moisture,  food  supply,  and 
the  environment  generally,  the  progenations  from  a  single  parent  cell 


a 


% 


FIG.  10. — Illustrating  zooglea  formation,  a,  bacillar  aggregates  resulting  from 
cohesion;  b,  aggregates  resulting  from  cohesion  of  bacilli  with  gelatinized  cell- walls;  c, 
streptococcus  formation  resulting  from  the  septation  of  a  coccus  form ;  d,  cohering  cocci 
forms;  e,  bacilli  united  end  to  end  (resulting  from  septation),  enclosed  in  a  gelatinous 
coat;/,  bacillar  thread  enclosed  in  gelatin;  g,  mycobacterial  form;  h,  irregular  cell  forms, 
as  Mycoderma  aceti. 

show  an  increasing  rate  of  septation,  a  stationary  period,  followed  by  a 
gradual  decline,  ending  in  total  cessation  of  all  septation,  and  in  death. 
These  life  cycles  have  not  yet  been  carefully  determined;  in  fact,  they  are 
but  little  understood.  It  is  highly  probable  that  the  cycles  of  existence 
play  a  very  important  part  in  the  course  and  development  of  diseases  of 
bacterial,  origin. 


GENERAL   MORPHOLOGY   AND   PHYSIOLOGY  57 

Whereas  the  period  from  one  septation  to  another  septation  is  very 
short,  the  life  cycle  referred  to  is  often  quite  long,  perhaps  months  and, 
under  certain  conditions  lasting  for  years.  The  period  of  the  life  cycle 
can  be  modified  artificially  by  food  supply,  chemicals,  etc. 

Investigators  have  succeeded  in  prolonging  the  life  cycle  oiParamecittm..— 
Normally  P.  caudatum  dies  out  in  about  175  generations;  but  by  applying 
alcohol  (1-5000  to  1-10,000)  the  cycle  has  been  increased  to  860  generations. 
Very  dilute  solutions  of  strychnine  gave  similar  results.  If  the  life  cycle  or 
vital  impulse  of  these  simple  organisms  can  be  prolonged  it  is  probable  that 
similar  effects  can  be  produced  in  higher  organisms.  Numerous  investi- 
gators have  from  time  to  time  sought  after  agents  which  might  inhibit  the 
senile  changes  in  cells  and  circulatory  system  (arteriosclerosis)  but  thus 
far  without  conclusive  results. 

Microbes  feed  upon  organic  substances  generally.  Those  which  feed 
upon  dead  organic  substances  are  said  to  be  saprophytic;  those  feeding 
upon  living  substances  are  said  to  be  parasitic.  If  they  can  live  on  dead 
organic  substances  only,  they  are  obligatively  saprophytic;  if  they  can  feed 
on  both  dead  and  living  organic  substances,  they  are  facultatively  sapro- 
phytic, or,  vice  versa,  facultatively  parasitic.  The  great  majority  of 
microbic  parasites  are  facultatively  so,  as  is  evidenced  by  the  fact  that 
they  can  be  grown  in  artificial  culture  media.  Many  of  the  microbic  sap- 
rophytes will  develop  on  living  substances  under  certain  conditions,  thus 
showing  that  they  are  facultatively  parasitic.  It  is  no  doubt  true  that  no 
known  microbic  parasite  actually  feeds  upon  the  living  substances  of  the 
various  hosts,  since  the  cytoplasm  is  in  all  instances  dead  before  it  is  taken 
up  and  assimilated  by  the  microbe.  It  would  therefore  be-  more  correct  to 
say  that  parasitic  microbes  are  biologically  associated  with  living  organ- 
isms, while  the  saprophytes  are  biologically  associated  with  dead  organic 
substances,  and  that  they  all  feed  upon  and  assimilate  dead  organic  sub- 
stances. In  certain  mutualistic  symbioses  (as  in  the  root  nodules  of  the 
Leguminosae)  the  biological  relationship  of  microbe  and  host  plant  is  very 
intimate,  but  there  is  no  actual  interchange  of  living  material. 

All  microbes  require  moisture  and  warmth  (comparatively  speaking) 
for  their  development,  although  they  are  enabled  to  withstand  greater  ex- 
tremes of  heat  and  cold  than  other  organisms.  The  temperature  of  liquid 
air  (about  —  27o°F.)  does  not  kill  them  at  once,  and  the  spores  may  be 
boiled  for  some  time  without  destroying  their  germinating  power.  Cold 
(freezing  temperature)  promptly  checks  growth  and  septation,  and  so  does 
dryness  and  excessive  warmth,  although  life  may  not  be  destroyed.  The 
majority  of  microbes  develop  most  actively  at  a  temperature  of  25°C., 
a  few  species  develop  more  actively  at  a  lower  temperature  (2o°C.),  and  a 
few  others  at  a  higher  temperature  (38°C.).  Those  which  develop  at  a 


58  PHARMACEUTICAL  BACTERIOLOGY 

temperature  ranging  from  o°C.  to  2o°C.  are  said  to  be  cold  loving  (psych  - 
rophile),  from  10°  to  45°C.,  mesophile,  from  40°  to  70°  C.,  thermophile. 
Thermophile  species  are  found  in  decaying  vegetable  matters,  whereas 
psychrophile  species  are  found  in  cold  water  and  cold  soils. 

Bacterial  life  processes  result  in  the  formation  of  many  substances, 
some  of  which  are  of  the  greatest  importance.  It  is  impossible  to  estimate 
properly  the  enormous  tasks  performed  by  these  minute  organisms,  nor 
shall  we  at  this  time  make  any  attempt  to  set  forth  the  great  good  and  the 
apparent  great  harm  done  by  them.  We  need  only  state  that  without 
rotting  microbes  soil  formation  woulji  be  impossible,  and  without  soil, 
higher  plant  and  animal  life,  as  we  now  know  them,  would  be  impossible. 
Without  plant  food  digesting  microbes  crop  growing  would  be  impossible. 
The  saltpeter  deposits  in  South  America  and  the  iron  deposits  of  the  Mesabi 
range  of  Minnesota  are  said  to  be  the  result  of  bacterial  action.  We  make 
extensive  practical  use  of  microbes  in  medical  practice,  in  the  dairying 
industry,  etc. 

We  will  mention  only  a  few  substances  of  undoubted  microbic  origin. 
Ptomaines  and  toxalbumins  are  well-known  poisons  elaborated  by  sapro- 
phytic  microbes  which  feed  on  meats  and  other  organic  substances,  causing 
the  familiar  putrefactive  changes.  Pahogenic  microbes  elaborate  toxins 
to  which  are  due  the  manifestations  of  the  disease.  Acetic  acid,  lactic  acid, 
and  butyric  acid  are  elaborated  by  Bacillus  aceticus,  B.  acidi  lactici,  and 
B.  butyricus,  respectively.  Some  species  liberate  odoriferous  substances, 
others  gases,  coloring  substances,  phosphorescence,  etc.  The  phosphores- 
cence observed  on  the  ocean  is  supposed  to  be  due  to  bacteria  (Bacillus 
phosphorescens  indicus).  Phosphorescent  bacteria  occur  in  dead  fish  and 
in  meat.  Old  cultures  in  animal  nutrient  media  and  in  the  presence  of 
sodium  salts  are  phosphorescent  in  the  dark,  sufficiently  so,  to  have  sug- 
gested making  bacterial  lamps  and  signal  lights. 

It  has  been  suggested  that  certain  diseases,  of  which  the  causes  are  at 
present  unknown  (as  yellow  fever,  measles) ,  may  be  due  to  organisms  so 
small  as  to  be  invisible  (ultra  micro-organisms).  It  is  known  that  the 
virus  of  yellow  fever  will  pass  through  the  most  compact  clay  or  porcelain 
filter.  Attempts  have  been  made  to  demonstrate  the  presence  of  ultra 
micro-organisms  by  special  photomicrographic  methods,  aided  by  special 
illuminating  devices  (the  ultra  microscope  of  Siedentopf  and  Szigmondy) 
but  without  success.  Furthermore,  no  one  has  succeeded  in  culturing 
such  theoretically  surmised  organisms  in  artificial  media,  which  would 
certainly  render  them  visible  en  masse.  It  may,  however,  be  possible 
that  some  ultra-organisms  are  obligative  parasites  hence  will  not  develop 
in  artificial  media. 

The  biological  (symbiotic)  relationship  of  different  species  of  bacteria 


GENERAL    MORPHOLOGY    AND    PHYSIOLOGY  59 

to  each  other  and  to  their  host  are,  in  many  instances  at  least,  not  well  under- 
stood. For  example,  it  is  not  clear  what  biological  relationship  the  dif- 
ferent species  of  bacteria  in  a  mixed  infection  bear  to  each  other.  In  the 
case  of  the  root  nodule  organisms  of  the  Leguminosae  it  is  known  that  there 
is  a  mutually  beneficial  (mutualistic  symbiosis,  mutualism)  relationship" 
between  microbe  and  host  but  it  is  not  obligatively  so,  since  the  symbionts 
can  exist  independently  of  each  other.  In  most  diseases  due  to  microbic 
invasion  there  is  one  species  of  bacterium  which  acts  as  the  primary  cause. 
It  is  known  that  tuberculosis,  especially  the  pneumonic  form,  usually 
shows  a  mixed  infection,  and  it  is  probable  that  the  associated  organisms 
as  bacteria  and  higher  fungi  act  as  predisposing  causes,  preparing  the 
tissues  so  as  to  yield  more  readily  to  the  invasion  of  the  primary  cause, 
the  Bacillus  tuberculosis.  Such  an  association  may  be  designated  com- 
pound symbiosis,  in  which  the  relationship  of  the  invading  organisms 
(secondary  and  primary)  is  mutualistic  and  the  relationship  of  these  to  the 
host  is  antagonistic.  It  is  known  that  certain  microbic  diseases  predispose 
to  other  microbic  invasions,  thus  we  may  say  that  these  organisms  are 
mutualistically  disposed  toward  each  other. 

Since  it  is  possible  to  cultivate  most  disease  germs  in  and  upon  artificial 
culture  media  (hence  dead  organic  substances)  it  is  evident  that  they  are 
only  facultatively  parasitic. 

In  many  instances  the  biological  association  of  bacteria  and  higher 
plants  and  animals  is  loosely  mutualistic,  as  the  bacteria  upon  roots  and 
rootlets  of  all  plants  and  the  bacteria  lining  the  intestinal  tract  of  animals. 
The  hay  bacillus  (Bacillus  subtilis)  is  a  constant  associate  with  the  Gra-. 
mineae  and  serves  an  important  function,  assimilating  or  binding  for  the  use 
of  the  host  plant,  the  free  nitrogen  of  the  air.  Certain  soil  organisms 
(Bacillus  megatherium,  B.  ellenbachiensis ,  B.  mesentericus,  B.  pyocyaneus, 
B.  prodigiosus,  the  Azotobacter  group,  Clostridium  pastorianum,  certain 
moulds  as  Aspergillus  niger  SindPenicillium  glaucum)  are  capable  of  assimi- 
lating the  free  nitrogen  of  the  air  thus  enriching  the  soil  for  the  benefit 
of  higher  plants. 


CHAPTER  V 
RANGE  AND  DISTRIBUTION  OF  MICROBES 

Microbes  are  omnipresent  over  the  surface  of  the  earth.  In  number 
and  in  bulk  they  exceed  all  other  organisms  (plants  and  animals)  put 
together.  They  form  a  large  percentage  of  the  bulk  of  the  soil.  They  occur 
in  the  air,  in  water,  in  snow,  in  hail,  in  raindrops,  in  and  upon  plants,  in 
and  upon  animals.  All  substances  with  which  we  come  in  contact  are 
likely  to  hold  microbes.  Our  clothing  teems  with  them.  They  are  in 
the  air  we  breathe,  in  the  food  we  eat,  and  in  the  liquids  we  drink.  The 
floating  dust  particles  of  the  air  carry  microbes;  the  particles  of  organic 
matter  in  water  harbor  microbes;  they  are  found  on  wood,  on  cloth,  on 
paper,  on  metal,  glass,  and  rock  surfaces,  in  fact  on  all  exposed  surfaces. 
The  hands,  the  hair,  the  entire  body  surface  of  man  and  of  the  lower 
animals  contain  or  hold  microbes.  They  line  all  mucous  membranes. 
The  mouth  cavity  is  a  veritable  bacteriological  laboratory.  The  entire 
intestinal  tract  teems  with  millions  upon  millions  of  these  minute  beings. 

Each  animal  and  each  plant  has  a  microbic  flora  peculiar  to  itself. 
Each  portion  of  the  plant  or  animal,  again,  has  distinctive  bacterial 
groups.  The  microbic  flora  of  the  intestinal  tract  of  the  dog  is  different 
from  that  of  the  pig,  or  cat,  or  fowl,  or  man.  Certain  species  predomi- 
nate in  the  mouth  cavity,  others  in  the  stomach,  still  others  in  the  small 
intestine,  in  large  intestine,  etc. 

Microbes  are  found  on  the  highest  mountain  peaks  and  in  the  deepest 
valleys.  It  is,  however,  true  that  the  higher  atmospheric  strata  contain 
fewer  microbes  than  the  lower  strata.  The  deeper  layers  of  soil  contain 
fewer  microbes  than  the  upper.  The  atmosphere  of  the  country  contains 
fewer  microbes  than  that  of  the  cities  and  towns.  Since  sunlight  and 
absence  of  moisture  are  natural  enemies  of  microbes,  we  may  expect  to  find 
microbes  more  abundant  in  dark,  damp,  and  moist  places  and  areas.  Mi- 
crobes are  always  more  abundant  in  cellars,  basements,  dark  hall- ways, 
and  alleys  than  they  are  in  attics,  sunlit  living  rooms,  and  along  broad 
boulevards  and  highways.  As  suggested  in  the  Chapter  on  the  Origin  of 
Bacteria,  cosmic  dust  or  telluric  and  interstellar  dust,  no  doubt  carry 
microscopic  organisms. 

Good  drinking  water,  whether  from  hydrant,  spring,  or  well,  contains 
only  a  comparatively  few  microbes,  from  fifty  to  one  hundred  per  cc.,  or 
even  less.  Stagnant,  foul  water  teems  with  microbes,  besides  other  organ - 

60 


RANGE   AND   DISTRIBUTION   OF  MICROBES  6 1 

isms,  such  as  protozoa.  So-called  pure  milk  contains  comparatively  more 
microbes  than  pure  water.  The  average  good  milk  contains  as  many 
as  30,000  microbes  per  cc.  Filthy  milk  may  contain  millions  of  microbes 
per  cc.  From  100,000  to  3,000,000  microbes  per  cc.  is  not  uncommon  in 
some  milk  which  careless  dairymen  declare  to  be  "good."  Soups,  broths, 
etc.,  boiled  squash,  potatoes,  meats,  and  cooked  organic  substances  gener- 
ally, if  allowed  to  stand  for  a  day  or  two,  contain  many  living  microbes. 
In  the  course  of  two  or  three  days,  if  the  weather  is  warm,  these  substances 
teem  with  microbes  and  are  rendered  wholly  unfit  for  human  consumption 
because  of  rotting  microbes  which  develop  highly  poisonous  ptomaines 
and  toxins. 

Microbes  do  not  grow  and  multiply  in  antiseptic  substances,  such  as 
strong  solutions  of  acids,  of  alkalies,  of  salts,  etc.  Used  and  dirty  cups, 
drinking  vessels,  milk  bottles,  dishes,  cooking  utensils,  knives,  spoons  and 
forks,  hold  numerous  microbes.  The  public  drinking  cup  has  been  the 
source  of  numerous  disease  infections.  Disease  is  carried  by  the  tools  of  the 
careless  dentist  and  by  the  clothing,  the  apparatus  and  the  clinical  thermom- 
eter of  the  indifferent  and  careless  physician.  The  hand-shaking  and  kiss- 
ing habits  spread  disease.  These  facts  are  generally  known  and  indicate 
the  wide  dissemination  of  the  different  kinds  of  microbes. 

From  the  foregoing  it  becomes  clear  that  microbes  are  present  almost 
everywhere,  and  that  it  is  impossible  to  escape  them.  It  is  the  aim  of  the 
science  of  bacteriology  to  distinguish  between  good  and  bad  microbes, 
between  those  which  are  desirable  and  those  which  are  undesirable,  be- 
tween useful  and  harmful  microbes.  It  is  not  the  aim  of  the  science  of 
bacteriology  to  destroy  them  all,  or  to  devise  ways  and  means  to  escape 
from  all  of  them.  In  fact,  we  owe  our  very  existence  to  these  very  minute 
organisms,  as  has  already  been  explained. 

Under  certain  conditions  bacteria  multiply  very  rapidly.  Such  sub- 
stances as  meat,  milk,  and  organic  foods  of  all  kinds,  if  exposed  to  moisture, 
warmth  and  removed  from  sunlight,  soon  swarm  with  microbes.  Certain 
non-pathogenic  microbes,  as  the  root  nodule  bacteria  (of  theLeguminosae), 
multiply  very  rapidly  within  the  tissue  cells.  Others  multiply  upon  the 
exterior  of  roots  and  of  root  hairs,  where  they  no  doubt  serve  a  useful  pur- 
pose to  the  plant.  In  bacterial  diseases  of  plants  and  animals  the  microbes 
multiply  very  rapidly  and  form  large  aggregates ,  as  a  rule .  To  pathological 
conditions  accompanied  by  extensive  and  general  bacterial  or  microbic  inva- 
sion, we  apply  the  term  bacteremia.  In  some  diseases  the  microbic 
invasion  remains  localized  and  yet  there  are  pronounced  general  or  sys- 
temic effects,  due  to  the  absorption,  into  the  system,  of  the  toxins  liberated 
by  the  microbes.  To  such  conditions  we  apply  the  term  toxemia.  Toxe- 
mia may,  however,  also  occur  in  bacteremia. 


62  PHARMACEUTICAL  BACTERIOLOGY 

Microbes  do  not  multiply  in  the  air  itself,  rather  upon  the  organic  dust 
particles  present,  provided  warmth  and  moisture  are  adequate. 

Since  microbes  multiply  rapidly,  perhaps  one  septation  in  from  twenty 
to  thirty  minutes,  it  is  evident  that  the  rate  of  numerical  increase,  under 
favorable  conditions,  is  very  great.  Allowing  thirty  minutes  for  each 
septation,  there  would  be  a  colony  of  2,097,152  microbes  in  ten  hours, 
developed  from  a  single  cell,  or  about  75,000,000,000,000  cells  in  twenty- 
four  hours.  However,  under  natural  conditions  septation  never  proceeds 
in  such  uniform  ratio.  All  manner  of  checks  to  septation  come  into  play 
sooner  or  later  which  may  finally  bring  about  complete  cessation  of  septa- 
tion and  sporulation. 


CHAPTER  VI 
BACTERIOLOGICAL  TECHNIC 

As  may  readily  be  supposed,  the  minuteness  and  wide  distribution  of 
microbes  call  for  special  methods  of  study  and  examination.  Even  the 
largest  forms  are  far  below  the  ken  of  unaided  vision.  Their  general  dis- 
semination through  organic  substances  calls  for  special  methods  for  the 
separation  and  isolation  of  individuals  or  single  bacterial  cells.  The 
difficulties  of  some  phases  of  laboratory  technic  are  further  increased  by 
the  resistance  of  spores  to  various  agents  and  substances  which  are  readily 
fatal  to  higher  organisms.  The  methods  of  examination  are  also  greatly 
complicated  by  the  marked  polymorphism  of  many  species. 

Bacteriological  technic  comprises  the  use  of  glassware,  compound 
microscope,  and  other  apparatus,  a  thorough  knowledge  of  sterilization  and 
disinfection,  the  preparation  and  use  of  culture  media,  the  making  of  micro- 


a  be 

FIG.  ii. — a,  Nest  of  beakers  and  reagent  bottle?.  The  smaller  and  medium  size 
beakers  are  more  desirable  for  bacteriological  work.  The  reagent  bottles  are  for  Canada 
balsam,  stains,  clearing  fluid,  etc. 

bic  cultures,  and  the  study  of  cultures.  Methods  vary  greatly.  The 
following  represents  a  brief  summary  of  general  methods  which  are  noted 
for  simplicity  and  which  have  proven  very  satisfactory  after  years  of 
testing. 

i.  Cleaning  the  Glassware 

All  glassware,  such  as  test-tubes,  flasks,  beakers,  Petri  dishes,  pipettes, 
shells,  bottles,  etc.,  which  is  to  be  used  in  bacteriological  work  must  be  dean; 
that  is,  free  from  all  extraneous  organic  as  well  as  inorganic  matter.  To 
accomplish  this,  it  is  necessary  to  use  an  abundance  of  pure  water,  hot  as 
well  as  cold,  aided  by  sand,  paper  shreds,  brushes,  towels,  alcohol,  acids, 
soap,  sodic  and  potassic  hydroxides,  and  whatever  else  may  be  necessary. 
Boil,  wash,  rinse,  and  wipe  within  and  without  repeatedly  until  it  looks, 

63 


PHARMACEUTICAL  BACTERIOLOGY 


and  is,  absolutely  clean.     The  following  solution  will  be  found  useful 
as  a  cleansing  agent  for  old  as  well  as  new  glassware : 


Potassium  Bichromate, 
Sulphuric  Acid, 
Water, 


6  parts. 
30  parts. 
40  parts. 


Of  course,  the  sulphuric  acid  must  be  added  little  by  little  with  constant 
stirring,  in  order  to  avoid  excessive  heat  development.     Soak  the  glassware 


FIG.  12. — Wire  baskets  for  holding  test-tubes.  Cylindrical  form  and  square  form. 
Each  basket  holds  about  fifty  test-tubes.  The  wire  is  galvanized  to  prevent  rusting 
The  round  wire  baskets  should  be  used. 

in  this  solution  for  some  time,  several  hours  or  more,  and  rinse,  wash,  drain 
and  wipe  thoroughly  afterward.  The  sole  object  to  be  attained  is  cleanli- 
ness in  the  true  sense  of  the  word.  The  glassware  must  be  clean  bacterio- 
logically  and  chemically;  that  is,  it  must  be  free  from  microbes  and 
chemical  substances. 

2.  Plugging  Containers  with  Cotton 

After  the  thorough  cleansing  above  outlined,  the  test-tubes  and  flasks 
are  plugged  with  a  good  quality  of  non-absorbent  commercial  cotton. 
The  dry  cotton  plug  forms  most  efficient  germ  filter.  All  microbes  are 
caught  and  held  in  the  meshes  of  the  cotton,  and  yet  the  air  is  permitted  to 
pass  through  into  the  tube  or  flask. 

Open  a  roll  of  cotton,  find  the  free  end,  and  lay  it  out  on  the  work  table. 
Take  the  test-tube  in  the  left  hand;  remove  a  goodly  tuft  of  cotton  with 
right  hand,  using  thumb  and  first  and  second  fingers.  Place  this  over 
the  mouth  of  the  tube  or  flask,  and  push  it  down  to  a  distance  of  J£  to  % 
inch  by  means  of  a  solid  glass  rod  rounded  (by  heat)  at  the  ends.  The 
rod  must  not  be  too  thick,  as  it  will  then  not  permit  enough  cotton  to  enter 
the  opening  nor  yet  too  thin,  as  it  will  then  be  forced  through  the  cotton. 
The  plug  must  not  be  too  tight,  as  that  would  interfere  with  subsequent 
manipulations  nor,  yet  too  loose,  for  obvious  reasons.  Enough  cotton 
should  project  above  the  opening  to  permit  of  ready  grasping  between  the 
fingers  in  the  later  operations. 


BACTERIOLOGICAL    TECHNIC 


Plugging  may  also  be  done  with  fingers  alone,  but  this  is  tedious  and 
non-professional.  A  far  better  method  is  to  use  a  pair  of  fairly  large 
blunt-pointed  pincers.  Remove  the  cotton  from  the  roll  by  means  of  the 
pincers  and  insert  it  into  the  test-tube  with  the  pincers. 


FIG.  13. — Wire  basket  filled  with  test-tubes  plugged  with  cotton.  A  little  cotton 
should  be  placed  in  the  bottom  of  the  basket  to  lessen  the  danger  of  breaking  the  test- 
tubes.  (Williams.') 


PIG.  14. — A  hot  air  sterilizer.  These  sterilizers  are  double-walled,  on  stand,  with 
perforations  at  top  for  thermometers.  Ordinary  baking  ovens  which  can  be  secured 
from  hardware  dealers  will  serve  the  purpose. 

Whatever  method  is  used,  remove  the  amount  of  cotton  required  to 
plug  one  tube  or  flask  at  one  time.  Do  not  attempt  to  plug  with  several 
small  pieces.  If  an  excess  of  cotton  projects  above  the  opening,  pluck 
it  away  with  the  fingers;  do  not  cut  it  away  with  scissors.  Plug  the  tubes 
as  uniformly  as  possible. 


66 


PHARMACEUTICAL  BACTERIOLOGY 


3.  Filling  Test-tubes  with  Culture  Media 

The  rule  is  to  pour  the  culture  media  hot,  although  this  is  not  abso- 
lutely essential.  For  example,  if  the  media  are  liquid  in  the  cool  or  cold 
state,  as  bouillon,  serum,  milk,  etc.,  they  may  be  poured  cold.  A  good 
rule  is  to  pour  a  desired  amount  of  the  media  just  as  soon  as  they  are 
prepared,  whether  they  are  still  hot  or  merely  warm  or  cold.  Of  course, 
gelatin  and  agar  media  must  be  poured  hot  or  must  be  liquefied  before 
they  can  be  poured. 

Fill  a  small  to  medium-sized  beaker  about  two-thirds  full  of  the  culture 
medium.  Grasp  a  plugged  tube  near  the  upper  end,  holding  it  between 
thumb  and  first  two  fingers  of  the  left  hand.  Remove  the  cotton  plug  by 


PIG.   15. — Diagrammatic  sectional  view  of  Arnold  steam  sterilizer  illustrating  the  prin- 
ciple of  steam  formation,  circulation  and  condensation. 

means  of  the  first  and  second,  second  and  third,  or  third  and  fourth  fingers 
of  the  right  hand,  grasping  the  free  portion  of  the  plug  with  the  back  of  the 
fingers  toward  the  cotton.  Holding  the  tube  slightly  inclined  on  a  level 
with  the  mouth,  take  beaker  with  medium  in  right  hand  (at  the  same  time 
holding  the  cotton  plug  as  described),  see  that  the  beak  rests  lightly  upon 
and  projects  slightly  over  the  edge  of  the  tube,  and  pour,  at  the  same  time 
shifting  the  eyes  to  the  lower  end  of  the  tube  to  watch  the  filling  process. 
Fill  tubes  one-third  full.  Set  down  the  beaker  and  replace  the  cotton 
plug.  Place  the  filled  tubes  in  special  wicker  baskets,  with  a  little  cotton 
at  the  bottom  to  prevent  breaking.  Some  practice  is  necessary  in  order  to 
pour  so  that  none  of  the  liquid  comes  in  contact  with  the  upper  third  of  the 
tube.  This  must  be  avoided,  in  order  to  prevent  the  cotton  plug  from 
sticking.  Tubes  may  also  be  filled  from  funnel  with  rubber  hose,  stop- 


BACTERIOLOGICAL  TECHNIC 


67 


cock,  and  glass  nib  attachment.  Occasionally  it  is  desirable  to  place 
exact  amounts  of  culture  media  in  the  tubes,  in  which  case  a  graduate,  a 
burette,  a  pipette,  or  other  convenient  measuring  device  may  be  used. 

4.  Sterilization  of  Culture  Media 

All  culture  media  in  tubes  as  above  set  forth,  and  the  portions  remain- 
ing after  the  desired  number  of  tubes  are  filled,  must  be  considered  as 
being  contaminated  with  living  microbes  and  their  spores.  These  mi- 
crobes and  spores  are  killed  by  the  sterilizing 
process.  For  all  ordinary  purposes  the  dis- 
continuous or  fractional  method  answers  the 
purpose  admirably.  Place  the  test-tubes, 
flasks,  and  other  cotton-plugged  containers 
with  culture  media,  in  a  steam  sterilizer 
(Arnold  steam  sterilizer,  either  board  of  health 
or  cylindrical  form ;  or  kitchen  vegetable  cooker 
or  steamer).  The  test-tubes  are  placed  in 
wire  baskets  (rectangular  or  cylindrical). 
These  several  containers  with  culture  media 
are  exposed  to  live  steam  for  about  thirty 
minutes,  whereupon  the  flame  is  turned  out, 
and  if  convenient  the  containers  are  allowed 
to  remain  in  the  sterilizer.  Caution  must  be 
observed  to  guard  against  condensed  steam 
running  into  the  several  containers.  The 
better  way  is  to  remove  the  containers  and 
place  them  in  an  incubator  kept  at  a  tem- 
perature of  20°  C.  In  twenty-four  hours,  or 
thereabouts,  steam  is  again  applied  for  thirty 
mnutes.  This  is  repeated  a  third  time  on 
the  second  day  after  the  first  sterilization. 
The  first  sterilization  presumably  kills  most  of 
the  vegetative  cells.  During  the  first  interval 
of  twenty-four  hours  most  of  the  spores  present 
develop  into  vegetative  cells,  which  are  killed 
at  the  second  sterilization.  Should  any  sur- 
vive the  second  steaming,  they  are  sure  to  be 
killed  during  the  third  sterilization.  During 
this  time  the  cotton  plugs  have  not  been 

removed.  The  media  thus  fractionally  or  discontinuously  sterilized  are 
now  ready  for  use  in  making  microbic  cultures,  or  they  may  be  set  aside 
for  an  indefinite  period  of  time. 


FIG.  i  6.  —  Autoclave  for 
using  steam  under  pressure 
for  purposes  of  sterilization. 
Many  different  forms  and  sizes 
of  autoclaves  are  on  the 
market.  Some  of  them  to  be 
used  with  gas  heat,  others 
with  electricity.  The  enor- 
mous autoclaves  used  by  the 
large  canneries  will  hold  sev- 
eral tons.  Enormous  sterili- 
zers on  the  order  of  the  auto- 
clave are  used  at  the  national 
quarantine  stations,  as  at 
Angel  Island,  San  Francisco, 
and  at  New  Orleans. 


68  PHARMACEUTICAL  BACTERIOLOGY 

It  is,  of  course,  evident  that  in  the  above  process  of  sterilization  the 
temperature  does  not  exceed  100°  C.,  and  it  maybe  less  in  certain  portions 
of  the  sterilizer,  steamer,  or  cooker,  say,  95°  to  97°  C.  Certain  kinds  of 
sterilizations  are  done  by  steam  under  pressure.  The  apparatus  used 
for  this  purpose  is  known  as  autoclave.  It  consists  of  a  strong  steam 
cylinder  with  a  screwed-down  top,  safety  valve,  steam  gauge,  and  ther- 
mometer. The  articles  (media,  etc.)  to  be  sterilized  are  placed  inside, 
the  top  is  securely  fastened  down,  steam  is  generated  until  the  thermometer 
registers,  say,  1 20°  C .  The  temperature  is  kept  up  to  that  degree  for  about 
10  to  20  minutes,  which  is  sufficient  to  destroy  all  life,  including  spores. 
For  certain  purposes  the  autoclave  is  not  applicable.  Blood  serum,  gelatin 
media,  and  all  media  containing  carbohydrates,  undergo  certain  chemical 
changes  when  the  temperature  is  raised  above  100°  C.,  or  even  if  kept  at 
100°  C.  for  a  long  time  or  for  a  short  time,  if  oft  repeated.  The  autoclave 
is  convenient  for  sterilizing  discarded  cultures,  test-tubes,  and  glassware 
generally,  and  such  media  as  beef  broth  and  agar. 

In  many  instances  it  is  desirable  to  sterilize  at  a  temperature  lower 
than  100°  C.  Albumen  and  blood  serum,  for  instance,  will  coagulate  at 
that  temperature.  Again,  it  is  desired  to  kill  the  microbes  without  de- 
stroying the  toxins  which  they  form,  as  in  the  manufacture  of  bacterial 
vaccines.  In  the  sterilization  (pasteurization)  of  milk,  a  lower  tempera- 
ture is  employed.  In  the  sterilization  of  these  and  other  substances  the 
temperature  ranges  from  50°  to  85°  C.  The  discontinuous  method  is 
employed,  differing  from  the  method  already  described  in  that  the  period 
of  exposure  is  much  prolonged,  about  one  hour.  The  number  of  daily 
exposures  ranges  from  one  to  six.  For  example,  milk  exposed  to  a  tem- 
perature of  60°  to  70°  C.  for  one  hour  is  considered  sufficiently  sterilized, 
whereas  blood  serum  is  subjected  to  hourly  exposures  of  a  temperature 
of  60°  C.  for  six  successive  days  before  it  is  pronounced  completely 
sterilized. 

5.  Preparation  of  Culture  Media 

The  pharmacist  should  give  especial  attention  to  the  preparation  of 
bacterial  culture  media,  as  in  this  he  may  be  of  service  to  the  physician. 
The  busy  general  practitioner  who  is  not  equipped  with  a  suitable  bacterio- 
logical laboratory,  or  who  does  not  have  time  to  prepare  culture  media, 
would  no  doubt  consider  it  a  very  decided  advantage  should  the  pharma- 
cist offer  to  assist  him.  This  will  be  more  fully  set  forth  in  the  last 
chapter. 

In  brief,  it  may  be  stated  that  microbes  feed  upon  the  same  substances 
that  we  feed  upon.  In  the  presence  of  adequate  warmth  and  moisture 
they  attack  all  organic  substances.  This  being  the  case,  it  may  readily 
be  assumed  that  there  are  many  substances  or  media  which  can  be  used 


BACTERIOLOGICAL   TECHNIC  OO/ 

as  food  for  bacteria.  Such  is  the  case,  and  the  number  of  media  which 
have  been  used  is  legion.  Almost  any  organic  substance  may  be  used, 
provided  it  is  not  antiseptic  in  its  properties. 

Culture  media  are  liquid  or  solid,  simple  or  compound.     In  the  case  of 
liquid  or  liquefiable  solid  media,  the  following  physical  properties  'are_ 
desired,  in  so  far  as  it  is  possible  to  attain  them : 

a.  Culture  media  should  be  perfectly  clear.  There  should  be  no  sedi- 
ment, no  opacity  or  flocculent  suspension,  and  no  floating  matter.  In  the 
case  of  broths,  extracts  generally,  gelatin  media,  and  blood  serum,  these 
requirements  are  easily  attained.  Perfectly  clear  agar  is  difficult  to- 
obtain.  Milk  is  normally  opaque. 


PIG.  17. — Arnold  Steam  Sterilizer.  Boston  Board  of  Health  Form.  This  sterilizer 
is  square,  and  constructed  with  a  side-door  all  in  accordance  with  the  recommendation 
of  the  Boston  Board  of  Health.  It  large  size  makes  it  well  suited  to  the  requirements  of 
.Board  of  Health  laboratories,  and  it  has  been  found  to  be  very  serviceable  and  conven- 
ient. It  is  made  of  copper  throughout,  following  the  same  principles  as  employed  in  the 
! Construction  of  the  other  sterilizers. 

b.  Media  should  be  neutral  or  very  slightly  alkaline  to  litmus,  which  is 
equivalent  to  a  slightly  acid  reaction  to  phenolphthalein,  at  a  temperature 
of  about  20°  C.    Most  microbes  develop  best  in  media  of  such  reaction. 

c.  They  must  be  free  from  living  microbes  and  their  spores,  and  from 
)ther  organisms.     This  requirement  is  attained  by  sterilization  as  already 
lescribed.     Culture  media  contaminated  with  living  organisms  are  not 
isable  in  bacteriological  work. 


70  PHARMACEUTICAL  BACTERIOLOGY 

The  essential  requirements  given  under  a,  b,  and  c  are  obtained  by 
filtration,  neutralization,  and  sterilization,  as  will  be  more  fully  explained. 
Non-liquefiable  solid  media,  as  potato,  bread,  squash,  etc.,  must  be  clean, 
free  from  living  microbes  and  other  organisms,  and  there  should  be  a 
comparatively  smooth  exposed  inoculating  surface.  These  requirements 
are  attained  by  washing  and  otherwise  cleansing,  disinfecting,  rinsing, 
and  heat  sterilization  (dry  heat,  steam  or  hot- water  bath). 


PIG.  18. — Arnold  Steam  and  Hot-Air  Sterilizer  for  Surgical  Instruments.  This 
sterilizer  is  a  combination  and  portable  sterilizer,  so  designed  that  instruments  may  be 
both  sterilized  and  then  dried  by  hot  air,  if  desired.  About  100°  C.  can  be  attained 
with  the  hot  air  by  simply  turning  the  valve  shown  in  the  illustration,  which  turns  the 
steam  as  it  escapes  from  the  chamber  into  the  base. 

The  following  are  the  more  important  media: 

A.  Nutrient  Bouillon. — 

Beef  Extract  (Armour's,  Liebig's,  etc.),  3  gin. 

Peptone,  10  gm. 

Salt,  5  gm. 

Distilled  Water,  1000  cc. 

Mix  ingredients  and  boil  for  a  few  minutes.  Filter  through  filter 
paper.  This  bouillon  may  be  modified  by  adding  glycerin  (6  per  cent.), 
and  sugars,  as  dextrose,  saccharose,  or  lactose  (i  per  cent.). 

B.  Loeffler's  Blood  Serum. — Very  largely  used  in  making  diagnostic 
diphtheria  bacillus  cultures.     In  many  cities  this  medium,  with  sterilized 
cotton  swabs,  in  sterilized  test-tubes,  is  furnished  free  to  physicians  by 
the  board  of  health.    In  cities  and  towns  where  this  is  not  done,  the 
pharmacist  should  be  prepared  to  furnish  the  materials  to  the  physicians. 
The  medium  consists  of  — 

Bouillon  with  i  per  cent.  Glucose,  i  part. 

Blood  Serum,  3  parts. 

The  bouillon  is  prepared  as  above  described,  with  i  per  cent,  of  glucose 
added.  The  blood  serum  can  be  obtained  from  calf,  sheep,  ox,  or  cow, 
through  the  butcher  or  at  the  abattoir.  Collect  the  blood  in  a  clean, 


BACTERIOLOGICAL   TECHNIC 


FIG.    19. — Cul- 
ture tube  and  swab 


sterile  jar  or  flask,  closed  with  cotton  plug.  Place  on  ice  for  twenty-four 
to  forty-eight  hours,  during  which  time  coagulation  has  taken  place;  the 
serum  may  then  be  siphoned  off.  The  proper  sterilization  of  Loeffler's 
serum  requires  care.  After  the  bouillon  and  serum 
are  mixed,  pour  into  test-tubes  and  coagulate  in  a 
Koch  serum  coagulator  at  a  temperature  of  80°  C. 
Any  form  of  sterilizer  may,  however,  be  used.  The 
essentials  are  that  the  temperature  should  be  raised 
very  gradually  and  must  be  kept  below  the  boiling- 
point,  and  the  tubes  should  be  slanted  at  a  degree 
which  will  bring  the  medium  close  to  the  cotton  plug, 
making  what  are  commonly  called  tube  slants.  After 
the  medium  is  coagulated  in  the  tubes  it  is  sterilized 
fractionally  on  three  successive  days  (one  hour  each 
day)  at  a  temperature  of  80°  C.  These  tube  slants 
are  now  ready  for  the  physician. 

To  prevent  evaporation  of  the  medium  in  the 
test-tubes,  cover  the  cotton  plug  and  upper  end  of 
tube  with  tin  foil  fastened  with  thread,  and  dip  into 

melted  paraffin  several  times.    Tubes  thus  sealed  can 
r  .  .  'iii 

be  kept  for  a  year  or  more  without  any  considerable    sicjans  in  the  diag- 

shrinking  of  the  medium.  Dip  the  tin  foil  in  a  i :  2000 
corrosive  sublimate  solution  before  capping  on  tubes. 
A  simpler  way  is  to  use  rubber  caps  which  are 
especially  made  to  fit  over  the  end  of  the  test-tube 
and  the  cotton  plug.  These  rubber  caps  must  be 
sterilized  before  applying  them,  for  which  purpose 
the  1-2000  corrosive  sublimate  solution  will  be  found  satisfactory. 
Rubber  stoppers  may  also  be  used  but  they  are  more  expensive  and 
inferior  to  the  rubber  cap  or  the  tin  foil  with  coat  of  paraffin. 

C.  Liquid  Blood  Serum. — Obtained  as  for  Loeffler's  serum.     Sterilize 
fractionally  at  a  temperature  of  from  56°  to  58°  C.  for  one  hour  on  each 
of  six  days.     The  serum  will  be  liquid  and  clear. 

D.  Milk. — Secure  fresh  milk  directly  from  cow,  or,  if  in  cities,  demand 
certified  milk.     Keep  on  ice,  in  a  covered  jar,  for  twenty-four  hours. 
Siphon  off  the  middle  portion,  rejecting  cream  and  sediment.     Sterilize 
like  Loeffler's  blood  serum.    Litmus  milk  is  prepared  by  adding  i  per  cent, 
of  azolitmin  before  sterilizing.    .This  indicator  will  show  whether  or  not 
acids  are  formed  by  the  microbes  which  may  be  cultivated  in  the  milk. 
Only  pure  milk  will  answer  the  purpose.     Milk  to  which  preservatives 
(formaldehyd,  salicylic  acid,  borax,  boric  acid)  have  been  added  must 
not  be  used. 


nosis  of  diphtheria. 
The  swab  tube 
should  be  long 
enough  to  have  the 
entire  length  of  swab 
inside,  not  project- 
ing as  shown  in  the 
figure.  (Williams.} 


72  PHARMACEUTICAL  BACTERIOLOGY 

E.  Peptone  Solution. — The  medium  is  employed  to  test  for  the  develop- 
ment of  indol  by  certain  bacteria.  It  consists  of 

Peptone,  10  gm. 

Salt,  /        5  gm- 

Distilled  Water,  1000  cc. 

Boil,  filter,  and  sterilize  as  for  bouillon.  The  bacteriological  indol  lest 
is  of  great  importance  in  medical  practice,  and  the  chances  are  that  physi- 
cians will  require  this  medium.  However,  sugar-free  beef  broth  is  also 
used  for  this  test;  in  fact,  it  is  generally  preferred.  Beef  contains  a  small 
amount  of  muscle  sugar,  which  must  first  be  removed. 


FIG.  20.  FIG.  21. 

FIG.  20. — Test-tube  cultures,  a,  Stab  culture.  Thifc  tube  isfclosed  with  a  rubber 
stopper  to  prevent  drying  of  medium;  b,  streak  or  smear  culture  on  slant,  tube  closed 
with  rubber  cap.  (Williams.) 

FIG.  21. — The  ordinary  rice  cooker.  A  most  valuable  apparatus  in  preparing  cul- 
ture media  and  for  sterilizing  test-tubes  and  other  objects. 

F.  Sugar-free  Bouillon. — Grind  the  fat-free  beef  through  a  meat 
grinder;  add  water,  and  inoculate  at  once  with  a  pure  culture  of  Bacillus 
coli  communis,  and  allow  to  incubate  for  twelve  to  fifteen  hours  at  38°  C., 
then  boil,  filter,  add  peptone  and  salt,  and  prepare  like  bouillon;  or, 
inoculate  nutrient  bouillon  with  the  colon  bacillus  and  prepare  as  above. 
However,  before  using  the  medium  it  should  be  tested  for  indol,  as  it  has 
been  proved  that  B.  coli  communis  may  form  indol  in  beef  extract.  The 
indol  test  in  bacterial  cultures  is  made  by  adding  two  drops  of  concentrated 


BACTERIOLOGICAL   TECHNIC 


73 


sulphuric  acid  and  one  drop  of  a  o.oi  per  cent,  sodium  nitrite  solution  to  a 
four-day  peptone-broth  culture.  If  a  pink  color  appears  at  the  end  of  one- 
half  hour  it  indicates  the  presence  of  indol. 

G.  Beef  Broth. — This  medium  is  now  not  as  extensively  used  as  £QT-_ 
merly.     It  is  more  difficult  to  prepare,  and  shows  no  advantages  over  the 
bouillon  already  described. 


Ground  or  Chopped  Lean  Beef, 

Peptone, 

Salt, 

Distilled  Water, 


500  gm. 

10  gm. 

5  gm- 

IOOO    CC. 


FIG.  22. — This  is  a  copper  double-walled  incubator  covered  with  non-conducting 
material  and  provided  with  a  water  gauge,  tabulations  for  thermometer  and  thermostat, 
a  ventilating  strip,  enclosed  base  and  inner  glass  door.  The  incubating  chamber  is 
24  cm.  high,  30  cm.  wide  and  24  cm.  deep. 

Add  the  water  to  the  minced  meat,  shake  frequently,  and  keep  on  ice 
for  twenty-four  hours,  then  strain  forcibly  through  cloth,  or  press  out  in  a 
hand  press.  Add  the  salt  to  the  liquid,  boil,  make  up  to  1000  cc.,  and 
add  the  peptone.  Titrate  to  reaction  of  +  i.o  per  cent.,  filter,  and 
sterilize.  It  will  be  apparent  that  the  cold  water  meat  infusion  con- 
tains merely  the  meat  salts,  meat  sugar,  and  acids,  and  a  certain  proportion 
of  the  albumens.  The  albumens  are  coagulated  and  removed  in  the 
filtering  process,  so  that  nothing  remains  of  the  meat  but  the  salts, 
acids,  and  the  trace  of  muscle  sugar.  Nearly  the  whole  of  the  meat 
proper  is  wasted.  It  is  apparent,  therefore,  that  the  meat  extract 
bouillon  answers  all  the  purposes  of  the  beef  broth. 


74 


PHARMACEUTICAL   BACTERIOLOGY 


H.     Gelatin  Medium. — 
Beef  Extract, 
Gelatin, 
Salt, 
Peptone, 
Distilled  Water, 


3  gm. 
100  gm. 

10  gm. 

IOOO    CC. 


Mix  ingredients  in  a  rice  cooker  and  boil  for  one-half  hour,  stirring  fre- 
quently; titrate  to  -f-i.o  per  cent,  and  filter.  This  forms  a  very  effi- 
cient culture  medium  for  most  bacteria,  and  is  clear  and  remains  solid 
at  ordinary  temperatures.  It  must  be  borne  in  mind,  however,  that 
frequent  or  prolonged  heating  tends  to  liquefy  gelatin  permanently. 


FIG.    23.  FIG.    24. 

FIG.  23. — Murrill's  Gas  Pressure  Regulator.  This  apparatus  in  its  most  improved 
form  is  to  be  used  in  connection  with  a  thermostat  for  the  maintenance  of  a  constant 
temperature.  The  use  of  this  regulator  relieves  the  thermostat  of  the  necessity  of  caring 
for  the  wide  variation  which  is  apt  to  occur  in  the  gas  pressure,  and  with  it  the  temper- 
ature may  be  held  constant  to  within  0.1°  C. 

FIG.  24. — Reichert  thermo-regulator  or  thermostat  used  with  incubator  and  other 
apparatus  requiring  a  uniform  degree  of  temperature.  May  be  used  in  conjunction  with 
the  gas  pressure  regulator. 

I.  Agar  Medium. — Agar  is  a  seaweed  found  on  the  Japanese  coast. 
It  forms  an  important  article  of  diet  among  the  Japanese  and  Chinese. 
The  medium  consists  of 

Beef  Extract,  3  gm. 

Agar,  15  gm. 

Salt,  5  gm. 

Peptone,  10  gm. 

Distilled  Water,  1000  cc. 


BACTERIOLOGICAL   TECHNIC  75 

Prepare  like  the  gelatin  medium.  Titrate  to  +1.0  per  cent. 
Agar  is  difficult  to  filter,  and  the  medium  is  never  quite  clear.  The 
agar  medium  liquefies  at  a  higher  temperature  than  gelatin,  and  does  not 
tend  to  remain  liquid,  no  matter  how  often  or  how  long  it  may~be 
heated. 

J.  A  gar-gelatin  Medium. — This  has  the  advantage  of  both  media,  and 
is  now  much  used  in  general  bacteriological  work. 

Agar,  8  gm. 

Gelatin,  40  gm. 

Salt,  5  gm. 

Peptone,  10  gm. 

Distilled  Water,  1000  cc. 

Mix,  boil  in  rice  cooker,  stir;  titrate  to  +  1.0  per  cent,  filter,  and 
sterilize  as  for  other  media. 

The  above  includes  the  more  important  culture  media  used  in  bacte- 
riological work.  Others  can  be  prepared  as  ocasion  requires.  It  is  not 
necessary  to  make  up  the  full  amounts  indicated  if  it  is  evident  that  smaller 
quantities  will  suffice.  The  student  should  prepare  all  of  the  media  in 
small  amounts  (one-quarter  the  quantities  given)  several  times,  in  order  to 
get  the  necessary  experience  and  practice. 

6.     General  Directions  for  the  Preparation  of  Culture  Media 

Book  information  alone  is  not  sufficient.  Experience  must  be  added. 
Also,  brief,  concise  explanations  are  far  more  valuable  than  lengthy  de- 
scriptions of  unessential  details.  Those  possessed  of  good  judgment  do  not 
require  lengthy  explanations,  and  lengthy  explanations  would  certainly  be 
wasted  on  those  who  lack  good  judgment.  This  does  not  imply,  however, 
that  it  is  unnecessary  to  adhere  strictly  to  established  methods.  The 
novice  must  follow  closely  the  methods  formulated  by  those  who  have 
devoted  many  years  to  some  one  particular  mode  of  procedure,  as  it  is 
wholly  unlikely  that  he  can  improve  upon  them.  Furthermore,  when  a 
physician  calls  for  Loeffler's  blood  serum,  for  example,  he  wishes  to  be 
assured  that  the  medium  has  been  prepared  according  to  the  standard 
method.  Any  substitution  or  deviation,  no  matter  how  slight,  may 
bring  about  wholly  negative  or  erroneous  results  and  conclusions.  With 
this  in  mind  the  following  suggestions  are  added: 

A.  Selection  of  Ingredients. — Great  care  must  be  observed  in  the  selec- 
tion of  the  ingredients  used  in  the  preparation  of  culture  media.  Meats 
used  must  be  from  healthy  animals,  and  there  must  be  absolute  certainty 
that  no  preservative  has  been  added.  Buy  the  meat  personally  from 
the  nearest  reliable  butcher  who  keeps  fresh  meats  only.  Remove  as 


76  PHARMACEUTICAL   BACTERIOLOGY 

much  of  the  fat  as  possible.     The  so-called  round  steak  of  beef  is  usually 
employed. 

Use  only  the  best  gelatin;  the  so-called  best  French  gelatin  is  usually 
employed,  although  much  of  the  " French  gelatin"  comes  from  Berlin, 
Chicago,  Omaha,  or  other  places  equally  remote  from  France.  Do  not 
attempt  to  use  old  friable  gelatin. 

The  milk  requirements  have  already  been  referred  to.  The  milk  must 
be  fresh,  placed  on  ice  at  once,  and  sterilized  within  twenty-four  hours 
after  it  is  taken  from  the  cow.  If  the  milk  is  obtained  from  an  unknown 
dealer,  test  it  for  the  presence  of  added  water,  preservatives,  and  other 
foreign  matter. 

Agar  does  not  deteriorate  readily,  and  may  be  kept  in  good  condition 
for  a  long  time.  Other  highly  gelatinous  seaweeds  may  be  used,  although 
this  is  not  permissible  in  the  preparation  of  any  of  the  standard  culture 
media  . 

'Serum,  egg  albumen,  peptone,  various  indicators,  etc.,  must  be  pure. 
Too  much  caution  cannot  be  observed  in  this  regard.  Secure  the  blood 
for  serum  personally  whenever  possible,  from  healthy  animals.  Use  egg 
albumen  from  fresh  eggs,  not  from  cold-storage  eggs  Dried  egg  albumen 
may  be  used.  Before  doing  so,  it  should  be  examined  microscopically 
and  if  it  contains  excessive  bacteria,  100,000,000  or  more  per  gram,  it 
should  not  be  used.  Much  of  the  dried  egg  and  dried  egg  albumin  of  the 
market  is  highly  contaminated  by  bacteria.  Peptone  and  other  chemicals 
should  be  secured  from  reliable  dealers. 

B.  Suggestions  on  the  Preparation  of  Culture  Media. — First  of  all,  some 
experience  is  necessary  before  a  neat  article  can  be  prepared.  Do  not 
expect  to  prepare  a  medium  which  meets  all  of  the  requirements  the  very 
first  time.  In  preparing  gelatin  media,  remember  that  these  are  injured 
by  excessive  heating,  and  in  preparing  agar  media,  remember  that  they 
are  very  difficult  to  filter.  Both  must  be  filtered  hot,  using  hot-water 
funnels;  or  the  ordinary  filtering  device  can  be  used  by  keeping  the  un- 
filtered  portion  hot  and  pouring  into  the  funnel  from  time  to  time.  Cover 
funnel  with  filter  paper  to  keep  out  dust,  and  keep  in  the  heat  as  much  as 
possible.  In  so  far  as  possible  filter  all  media  through  filter  paper  (one 
thickness,  properly  folded),  but  it  is  practically  impossible  (for  reasons  of 
time)  to  pass  agar  through  filter  paper.  This  medium  is  usually  filtered 
through  cotton  upon  which  a  neatly  folded  and  perforated  sheet  of  filter 
paper  has  been  placed.  Puncture  the  filter  paper  several  times  with  a 
small  knife  blade.  Filtering  through  cotton  is  quick,  but  the  media  are 
much  less  clear  than  when  filtered  through  filter  paper.  The  filtering 
process  may  also  be  hastened  by  means  of  pressure  (suction) ;  connect  fun- 
nel with  aspirator  bottle  and  pump,  but  see  to  it  that  the  connections 


BACTERIOLOGICAL    TECHNIC  77 

with  the  hydrant  are  properly  made  and  that  the  flow  is  properly  regu- 
lated, in  order  to  guard  against  any  back  pressure,  which  may  cause  the 
receiver  to  fill  with  hydrant  water.  This  accident  is  best  avoided  by 
interpolating  a  flask  or  bottle.  Agar  may  also  be  clarified  by  precipita- 
tion. Pour  the  hot  agar  into  an  ordinary  percolator  used  by  pharmacists. 
The  dirt  particles  and  other  impurities  will  gradually  settle  to  the  bottom. 
When  cool,  take  out  the  solid  medium  and  cut  away  the  lower  portion 
containing  the  sediment. 

C.  Titration  of  Culture  Media. — As  already  stated,  most  bacteria 
grow  best  in  neutral  or  very  slightly  alkaline  (to  litmus)  media,  and  since 
most  media  are  quite  decidedly  acid  in  reaction,  it  is  desirable  to  alkalinize. 
This  is  done  by  means  of  normal  sodium  hydroxide  solution.  In  order  to 
understand  the  method  of  procedure  clearly,  it  is  necessary  to  make  certain 
explanations. 

A  normal  (N/i) solution  of  any  substance  contains  as  many  grams  per 
liter  of  the  substance  as  there  are  units  in  its  molecular  weight,  if  the  sub- 
stance contains  one  atom  of  replaceable  hydrogen.  If  it  contains  two  atoms 
of  replaceable  hydrogen,  the  number  of  grams  used  equals  the  molecular 
weight  divided  by  two,  and  so  on.  According  to  this,  a  normal  solution  of 
sodium  hydroxide  contains  40  gm.  of  sodium  hydroxide  in  a  liter.  Exact 
normal  solutions  are,  however,  not  prepared  by  weight.  Crystallized 
oxalic  acid  is  used  as  the  basis  for  making  normal  solutions.  This  acid 
has  a  molecular  weight  (including  a  molecule  of  water  of  crystallization) 
of  126,  and,  since  it  is  dibasic,  63  gm.  per  liter  are  taken.  Any  normal 
acid  solution  will  exactly  neutralize  an  equal  volume  of  normal  alkaline 
solution.  To  make  a  normal  sodium  hydroxide  solution,  add  about 
14  gm.  of  pure  caustic  soda  to  one  liter  of  distilled  water.  Determine  the 
amount  of  this  solution  required  to  just  neutralize  i  cc.  of  normal  oxalic 
acid  solution.  This  volume  contains  the  quantity  of  sodium  hydroxide 
which  should  be  present  in  i  cc.  of  normal  solution,  and  from  this  we  may 
calculate  the  volume  of  distilled  water  to  be  added  in  order  that  i  cc.  of 
sodium  hydroxide  solution  will  neutralize  i  cc.  of  normal  oxalic  acid  solu- 
tion. Having  a  normal  solution  of  sodium  hydroxide,  it  is  now  possible  to 
prepare  a  normal  solution  of  hydrochloric  acid,  etc.  A  tenth-  (N/io), 
twentieth-  (N/2o),  fiftieth-  (N/5o)  normal  solution  is  a  normal  solution 
diluted  ten,  twenty,  and  fifty  times. 

An  acid  reaction  is  indicated  by  +,  and  an  alkaline  by  — .  The  degree 
of  acidity  of  any  culture  medium  in  preparation  may  be  indicated  by  the 
amount  of  normal  sodium  hydroxide  solution  required  to  render  it  neutral 
to  phenolphthalein.  Neutralization  by  titration  is  done  as  follows: 
Place  5  cc.  of  the  medium  to  be  neutralized  in  a  dish,  add  45  cc.  of  distilled 
water,  stir,  and  bring  to  a  boil.  Add  i  cc.  of  phenolphthalein  solution 


78  PHARMACEUTICAL  BACTERIOLOGY 

(0.5  per  cent,  of  phenolphthalein  in  50  per  cent,  alcohol).  Add  enough 
of  twentieth-normal  sodium  hydroxide  solution  (in  a  burette) ,  with  constant 
stirring,  to  give  a  faint  but  distinct  pink  color.  Read  the  amount  of  twen- 
tieth-normal sodium  hydroxide  necessary  to  neutralize  the  5  cc.  of  medium 
and  from  this  calculate  the  amount  of  normal  sodium  hydroxide  solution 
necessary  to  neutralize  the  entire  quantity  of  culture  medium.  Now  boil 
the  medium  and  again  titrate,  when  it  will  be  found  that  there  is  a  slight 
acid  reaction.  A  third  titration  is  rarely  necessary. 

Another  method  is  to  take  10  cc.  of  the  culture  medium,  add  a  few  drops 
of  the  phenolphthalein  solution.  From  a  burette  add,  drop  by  drop,  with 
constant  stirring,  a  normal  sodium  hydroxide  solution  (0.4  per  cent.)  until 
a  faint  pink  color  appears,  which  indicates  the  beginning  of  the  alkaline  reac- 
tion. Repeat  this  with  two  more  samples.  Note  the  amount  of  sodium 
hydroxide  solution  required  in  each  case,  and  take  the  average  and  calcu- 
late the  amount  required  for  the  entire  quantity  of  medium.  If,  for  exam- 
ple, the  average  was  i  cc.  for  each  10  cc.  of  medium,  then  1000  cc.  of  bouillon 
would  require  100  cc.  of  the  sodium  hydroxide  solution;  a  concentrated 
solution  being  used,  in  order  to  avoid  the  dilution  of  the  medium  with  the 
water  of  the  caustic-soda  solution.  Flocculency  of  the  medium  usually 
indicates  excessive  alkalinity. 

,  The  old,  crude,  rough-and-ready  method  is  to  add,  from  a  beaker, 
drop  by  drop,  a  tenth-normal  sodium  hydroxide  solution,  with  constant 
stirring,  until  red  litmus  paper  just  begins  to  turn  blue.  In  practice  it  is 
found  that  when  a  culture  medium  is  neutral  or  slightly  alkaline  to  litmus 
it  is  still  acid  to  phenolphthalein.  In  fact,  it  is  claimed  that  most  bacteria 
develop  best  in  a  medium  having  a  reaction  indicated  by  +i  or  +0.5 
that  is,  it  is  sufficiently  acid  to  phenolphthalein  to  require  i  per  cent,  or  0.5 
per  cent,  of  normal  sodium  hydroxide  solution  to  render  it  neutral  to 
phenolphthalein . 

D.  Suggestions  on  the  Preparation  of  Culture  Media  for  Physicians. — First 
of  all,  the  pharmacist  must  have  the  necessary  laboratory  equipment  and 
necessary  skill  and  experience  to  prepare  culture  media.  He  should  ex- 
plain to  a  few  representative  physicians  that  he  is  ready  to  prepare  such 
media  as  the  busy  physician  may  require.  The  physicians  will  in  all  proba- 
bility indicate  that  media  are  likely  to  be  needed  in  the  course  of  their 
practice.  Allow  yourself  to  be  guided  by  these  several  suggestions  and 
prepare  the  media  accordingly. 

Make  sure  that  the  culture  media  are  clear.  There  must  be  no  sedi- 
ment and  no  flocculency.  Not  infrequently  the  medium  fails  to  become 
sufficiently  clear,  even  though  every  precaution  has  been  taken.  In  such 
cases  clarification  may  be  tried,  rather  than  to  discard  it.  Add  the  white 
of  an  egg,  thoroughly  beaten,  to  a  liter  of  the  medium  in  the  liquid  state 


BACTEKIOLOGICAL   TECHNIC 


79 


and  at  a  temperature  below  the  coagulating  point  for  albumen,  mix  thor- 
oughly; boil  for  ten  minutes,  and  filter.  The  coagulating  albumen 
takes  up  the  impurities  which  remain  upon  the  filter  with  the  albumen, 
while  the  medium  comes  through  perfectly  clear. 
Media  which  have  become  infected  with  bacteria  as 
the  result  of  inadequate  sterilization  should  be  dis- 
carded. Do  not  attempt  to  clarify  them.  They  may 
become  clear,  but  they  are  nevertheless  objectionable 
because  of  the  substances  which  the  bacteria  may 
have  liberated  and  which  might  interfere  with  the 
development  of  the  bacteria  to  be  grown  in  it 
subsequently. 

Most  of  the  tubes  with  solid  media  (Loeffler's 
serum,  gelatin,  agar,  and  gelatine-agar)   should  be 
slants.    The  slanting  surface  offers  certain  advan-   flame  be  blown  out, 
tages  in  making  diagnostic  bacterial  cultures.    The   shuts^ff^he^as^ 
usual,  non-slanting   tubes,  for   deep  stab  cultures, 
should,  however,  also  be  held  in  readiness.     Keep  all  tubes  in  suitable  con- 
tainers, in  a  dry,  cool,  clean  place.    To  guard  against  infection  by  mold 
and  other  organisms,  it  is  well  to  cap  all  tubes  with  the  rubber  caps  or  the 


FIG.    26. — Hot  water  funnel  with  stand  and 
ring  gas  burner. 


FIG.  27. — Hot  water  funnel  with 
stand. 


tin  foil  dipped  in  corrosive  sublimate  and  paraffin,  as  already  suggested. 
In  case  of  liquid  media,  the  rubber  stoppers  or  the  rubber  caps  are  much 
preferred,  or  the  hot  paraffin  may  be  painted  over  the  tin  foil  and  upper 


8o 


PHARMACEUTICAL   BACTERIOLOGY 


end  of  tube  by  means  of  a  small  brush.     Apply  two  or  three  coats.     Thus 
protected,  there  is  no  danger  of  outside  infection. 

The  chances  are  that  the  physician  who  calls  for  tube  culture  media 
will  also  require  the  use  of  an  incubator.     This  the  pharmacist  shouk 


FIG.   28. — Glass  rods  with  platinum  wire,  straight  and  loop,  for  inoculating  culture  tubes 
Petri  plates,  etc. — (Williams.) 

have  in  readiness.  The  usual  copper  double-walled  water-jacket  incuba 
tor,  with  thermo-regulator,  kept  at  a  temperature  of  about  37°  C.,  wil 
serve  the  purpose. 

The  swab  to  be  supplied  with  each  tube  of  slanted  Loeffler's  serum 
consists  of  a  piece  of  wire  or  of  pine  wood  four  inches  long,  around  the 


a  b  c 

FIG.   29. — Cover-glass  pincers,     a  and  6  are  self-clamping  but  the  pressure  is  often 
enough  to  break  thin  covers. 

lower  end  of  which  a  pledget  of  absorbent  cotton  has  been  wound  and 
firmly  tied  by  means  of  thread.  This  is  placed  in  a  test-tube,  which  is  then 
plugged  with  cotton  and  sterilized  in  the  dry  sterilizer  (one  hour  at  a  tem- 
perature of  1 50°  C.) .  The  physician  wipes  the  cotton  end  of  the  swab  over 


BACTERIOLOGICAL    TECHNIC 


8l 


the  suspected  throat  area,  and  then  lightly  rubs  it  over  the  surface  of  the 
serum  tube  slant.  The  swab  is  returned  to  the  tube,  the  cotton  plug  is  re- 
stored and  then  returned  to  the  board  of  health  to  be  destroyed  in  stove 
or  furnace  fire,  or  destroyed  by  the  attending  physician  in  case  there  is  no 
board  of  health  to  receive  it. 

7.  Making  Bacterial  Cultures 

This  branch  of  the  science  of  bacteriology  is  of  comparatively  little 
importance  to  the  pharmacist.  While  it  is  desirable  to  know  what  bacterial 
cultures  are  and  how  to  make  some  of  them,  it  is  wholly  unlikely  that  the 
pharmacist  will  be  called  upon  to  do  extensive  work  along  this  line.  This 
is  the  work  of  those  who  make  bacteriology  a  specialty. 
Such  bacterial  cultures  as  are  likely  to  come  to  the  notice 
of  pharmacists  will  most  generally  be  prepared  by  phy- 
sicians, health  officers,  and  other  specialists  in  bacteriology. 
The  pharmaceutical  bacteriologist  may  be  called  upon  to 
make  bacterial  examinations  of  drinking  water,  of  milk, 
of  ice  cream,  and  other  food  materials;  of  syrups,  liquors, 
aquae,  tinctures,  fluidextracts,  infusions,  etc.,  and  he 
should,  if  possessed  of  some  skill  and  adequate  labora- 
tory facilities,  be  able  to  do  so. 

The  prime  object  in  growing  bacteria  in  artificial 
culture  media  is  to  make  possible  their  further  more  care- 
ful and  more  extended  study.  The  study  of  bacteria  in 
their  natural  or  normal  surroundings  is  all-important, 
but  is  not  complete  without  the  artificial  culturing. 

As    a  rule,  bacteria   are  biologically  associated  with 
other  organisms,  and  it  is  unusual  to  find  pure  cultures 
in  nature  or  in  natural  media.     An  open  sore  may  contain 
several  or  many  species  and  varieties  of  bacteria,  in  addi- 
tion to  the  pus  germs.     The  intestinal  tract  of  the  cholera 
patient  contains  bacteria  other  than  the  comma  bacillus 
of    Koch.     The   tubercular  bronchials    always    show    a 
mixed    infection.     The    diphtheric    membrane    contains 
some  foreign  germs,  etc.     Some  infections,  particularly 
those  of  internal  tissues  or  organs,  as  lymphatic  glands    in  the  bottom  of 
for   example,    may   present    practically    pure    cultures.    ^**be'    ' 
However,   no  matter   how  mixed  an  infection  may  be, 
there  is  always  a  predominating  type  present,  or,  to  state  it  more  cor- 
rectly, it  is  the  greater  development  of  the  predominating  type  which 
determines  the  diagnostic  characteristics  of  the  infection. 

It  must  also  be  borne  in  mind  that  bacteria  behave  differently  when 


FIG.  30. — Cotton 
plugged  tube 
with  a  potato 
slant  res  ting  en  a 
bit  of  glass  rod  to 


82 


PHARMACEUTICAL  BACTERIOLOGY 


taken  out  of  their  natural  environment  and  placed  in  artificial  culture 
media.  It  does  not  at  all  follow  that,  in  the  case  of  a  mixed  infection, 
the  predominating  and  diagnostic  microbe  will  remain  the  predominat- 
ing type  when  said  mixed  infection  is  transferred  to  some  artificial 
culture  medium.  In  fact,  the  predominating  microbe  may  develop 


FIG.  31. — Manner  of  holding  tubes  when  making  subcultures.  The  cotton  plugs, 
removed  from  the  two  tubes, .  hould  be  held  in  hand  holding  the  platinum  rod,  as  explained 
in  the  text.  (In  this  figure  the  cotton  plugs  are  held  in  the  hand  holding  the  test 
tubes,  which  is  wrong.)  (Williams.) 

very  slowly  or  with  great  difficulty,  if  at  all,  in  the  artificial  culture 
media;  whereas  one  or  more  of  the  associated  microbes  may  thrive 
remarkably  well,  soon  entirely  overshadowing  the  former.  These  and 


FIG.  32. — Making  an  E?march  roll-tube  culture.  A  lump  of  ice  is  placed  in  a  dish 
and  the  inoculated  tube  is  placed  horizontally  in  a  groove  in  the  ice  and  revolved  until  the 
medium  is  well  set.  The  groove  may  be  made  with  test-tube  full  of  hot  water.  (Williams.) 

other  conditions  occasion  some  of  the  great  difficulties  encountered  in 
determining  the  primary  causes  of  some  microbic  and  protozoic  diseases 
and  infections. 

A.  Test-tube  Cultures. — Inoculate  several  test-tubes,  containing 
nutrient  gelatin  or  agar  gelatin,  with  any  material  which  is  known  to 
be  bacterially  infected.  This  is  done  by  touching  the  infected  material 


BACTERIOLOGICAL   TECHNIC 


with  the  tip  of  a  heat-sterilized  (by  holding  in  flame  of  Bunsen  burner  until 
red  hot)  platinum  needle  (prepared  by  fusing  a  platinum  wire,  iK  Inches 
long,  into  the  end  of  a  glass  rod,  six  to  seven  inches  long),  then  removing 
the  cotton  plug  from  the  test-tube,  and  pushing  the  needle,  carrying  the 
microbes,  into  the  culture  medium  down  to  the  very  bottom  of  the  tube. 
Replace  the  cotton  plug  at  once,  pass  the  needle  into  the  flame  of  the  Bunsen 
burner  until  red  hot,  to  sterilize  it,  and  lay  aside  for  the  next  tube  inocu- 
lation. This  is  known  as  a  deep  stab  tube  inoculation.  In  this  manner 
inoculate  some  five  or  six  tubes.  Also  make  streak 
inoculation  on  tube  slants  by  simply  passing  the  in- 
fected platinum  needle  over  the  middle  of  the  tube 
slant  surface,  from  lower  end  toward  the  top,  ob- 
serving the  instructions  regarding  the  cotton  plug 
and  needle  sterilization,  with  each  tube  inoculation. 
Number  the  tubes  serially,  and  in  a  special  note- 
book make  entry  of  all  desirable  data  pertaining 
to  each  inoculation,  making  such  entries  under 
each  tube  number.  Place  tubes  vertically  in  a 
suitable  holder,  as  tumbler,  beaker,  wire  basket, 
etc.,  and  set  aside  in  incubator  or  in  some  container 
to  which  you  alone  have  access. 

In  warm  weather  the  first  bacterial  growths  may 
appear  at  the  end  of  thirty-six  hours.  In  cold  or 
cool  weather  nothing  may  appear  for  two,  three, 
and  even  four  to  five  days.  Note  the  nature  of  the 
bacterial  growth  in  a  deep  stab  inoculation  and  in 
the  streak  inoculation,  as  to 

a.  Growth — scanty,  moderate,  abundant;  slow, 
rapid. 

b.  Form    of    growth — outline    clearly    defined, 
spreading,  rugose,  beaded,  etc. 

c.  As  to  surface — flat,  raised,  concave,  convex. 

d.  Color — translucent,   glistening,  waxy,  trans- 
parent, opaque,  light,  chalky  white,  grayish-white, 


FIG.  33. — Kitasato 
filter  for  filtering  hypo- 
dermicsolutions,  culture 
media,  sera,  water,  etc. 
The  material  to  be  filt- 
ered is  placed  in  the 
globose  container  and 
forced  through  the  clay 
(infusorial  earth)  tube 
(Berkefeld  filter  bougie) 
by  connecting  the  re- 
ceiver with  a  vacuum 
pump.  All  parts  of  the 
filter  must,  of  course,  be 

dark  red,  green,  blue,  yellow,  lemon  color,  purple,  sterilized  by  heat  be- 
fore and  after  using. 
(Williams.) 


etc. 


e.  Odor — comparative  description. 

f .  Consistency — viscid,  slimy,  stringy,  membranous,  friable  or  brittle, 
dry,  watery,  etc. 

g.  Changes    in    medium — gelatin    liquefied,    gelatin    not    liquefied; 
colored,  as  grayed,  browned,  reddened,  blued,  etc.     In  case  indicators 
are  used,  any  color  changes  should  be  noted. 


84 


PHARMACEUTICAL   BACTERIOLOGY 


h.  Deep  stab  culture — where  is  growth  most  active?  If  at  bottom, 
it  indicates  anaerobic  tendencies.  If  limited  to  top  of  medium,  it  indi- 
cates decidedly  aerobic  tendencies.  (Most  bacteria  are  decidedly  aerobic; 
that  is,  they  require  free  oxygen  to  thrive.) 

The  test-tube  cultures  do  not  necessarily  represent  pure  cultures, 
and  the  student  cannot  know  whether  the  growths  in  the  test-tubes 
represent  the  predominating  bacterial  flora  in  the  substance  from  which 
the  inoculations  were  made.  The  chief  object  in  making  the  above 


FIG.  34. — Streak  culture  on  agar  in  a  Petri  dish.      (Delafield  and  Prudden.) 

cultures  is  to  enable  the  student  to  get  practice  in  this  preliminary  work, 
particularly  as  to  making  the  cultural  observations  above  indicated. 

The  student  should  now  make  transfers  (sub-cultures)  from  the  first 
tube  cultures  into  second  tubes,  and  note  whether  or  not  the  charac- 
teristics originally  noted  are  continued  or  repeated.  If  the  transfer 
cultures  are  the  same  as  the  originals,  it  is  an  indication  that  the  first 
cultures  were  pure  (representing  one  species  or  variety),  which  is  gen- 
erally the  case,  though  it  must  be  borne  in  mind  that  one  and  the  same 


BACTERIOLOGICAL    TECHNIC  85 

species  of  microbe  may  undergo  considerable  change  in  extended  cultur- 
ing,  as  indicated  in  the  changed  culture  characters.  In  fact,  some  of  the 
changes  are  so  extreme  as  to  confuse  even  the  most  expert  bacteriologists. 
B.  Isolating  Bacteria  by  the  Plate  Method. — In  order  to  separate 
or  isolate  the  several  species  and  varieties  of  bacteria  in  any  con- 
taminated substance,  it  is  only  necessary  to  dilute  the  inoculating 
material  sufficiently.  For  this  purpose  there  is  necessary,  sterilized 
Petri  dishes  containing  heat-sterilized  gelatin  or  other  solid  media  through 
which  the  bacteria  from  the  contaminated  substance  are  disseminated 
in  numbers  so  small  that  the  colonies  from  each  and  every  microbe 
present  may  be  visible  to  the  naked  eye  (or  aided  by  the  microscope). 
This  is  done  as  follows : 


FIG.  35. — Appearance  of  colonies  on  gelatin  in  a  Petri  dish.  Differences  in  size  of 
colonies  may  indicate  different  species.  Differences  in  color  also  indicating  different 
species,  cannot  be  shown  in  the  figure.  (Williams.} 

To  obtain  isolation  cultures  of  air  bacteria  it  is  only  necessary  to  expose 
the  Petri  dish  (with  a  layer  of  gelatin  or  agar-gelatin  medium,  sterilized) 
for  about  two  minutes,  immediately  closing  the  dish  and  setting  it  aside 
to  await  developments.  Making  isolation  cultures  from  contaminated 
solids  or  liquids  is  not  quite  so  simple.  Proceed  as  follows:  Liquefy  the 
gelatin  in  four  or  five  test-tubes  and  keep  them  at  a  temperature  of  not 
more  than  30°  C.,  just  high  enough  to  keep  the  contents  liquid;  set  them  in 
a  beaker  filled  with  warm  water  (30°  C.)  until  needed.  Number  the  tubes 
from  i  to  5. 

Dip  a  platinum  loop  (bend  the  end  of  a  straight  needle  into  a  small 
loop)  into  the  infected  liquid,  as  bouillon,  milk,  water,  tea,  syrup,  tincture, 
fluidextract,  etc.,  etc.,  and  pass  one  loopful  into  tube  No.  i  (sterilize 


86  PHARMACEUTICAL  BACTERIOLOGY 

loop  and  return  to  its  proper  place).  Rotate  tube  (replugged  with  the 
cotton  and  held  vertically)  rapidly  between  the  hands  for  twenty  seconds, 
to  mix  contents.  By  means  of  the  platinum  loop  take  two  loopfuls  (one 
loopful  may  serve)  from  tube  No.  i  (which  you  have  just  inoculated  and 
rotated)  and  pass  them  into  tube  No.  2.  Plug  both  tubes,  set  aside  tube 
No.  i,  and  rapidly  rotate  tube  No.  2.  Take  two  loopfuls  from  tube  No.  2 
and  transfer  to  tube  No.  3,  and  proceed  as  before.  Now  pour  contents 
of  tube  No.  i  into  a  sterile  Petri  dish,  also  numbered  i;  contents  of  tube  2 
into  Petri  dish  2;  and  tube  3  into  Petri  dish  3.  Wait  until  the  media  in 
the  Petri  dishes  are  solidified,  and  then  set  aside  at  the  room  temperature 
to  await  developments.  In  the  course  of  two  or  three  days  it  will  perhaps 
be  found  that  very  many  minute  specks  are  visible  in  dish  No.  i,  some  one 
hundred  or  more  may  appear  in  dish  No.  2,  and  perhaps  not  more  than  ten 
or  twenty  in  dish  No.  3.  Observe  carefully  the  several  growths  in  dishes 
2  and  3.  Each  visible  growth  indicates  the  development  from  a  single 
microbe.  Are  the  several  growths  all  alike,  or  do  they  differ?  .Differences 
in  color  and  in  outline  of  growths  indicate  different  species  of  bacteria. 
The  several  different  kinds  of  bacteria  may  now  be  transferred  to  test- 
tubes  by  means  of  the  straight  platinum  needle  or  the  loop,  and  the 
observations  may  thus  be  extended.  Transfers  can  be  made  to  different 
kinds  of  media,  as  agar,  gelatin,  agar-gelatin,  beef  broth,  milk,  prepared 
potato,  etc. 

C.  Making  Bacterial  Counts. — In  order  to  determine  the  number  of  bac- 
teria in  any  given  substance,  the  same  procedure  as  was  just  described  is  f  ol- 


FIG.  36. — Petri  dish.     These  dishes  are  among  the  essentials  in  the  bacteriological 

laboratory.     (Williams.') 

lowed,  with  the  difference  that  a  definite  amount  of  the  thoroughly  mixed 
contaminated  substance  is  added  to  a  definite  amount  of  culture  medium  in 
the  test-tubes  in  which  the  dilution  mixtures  are  made.  For  example,  we 
will  suppose  that  it  is  desired  to  determine  the  number  of  bacteria  (per  cc.) 
in  milk:  Thoroughly  mix  the  sample  of  milk  by  shaking  it  in  the  container. 
Take  o.i,  0.2,  0.5,  or  i  cc.  of  the  milk  (by  means  of  a  sterilized  graduated 
pipette)  and  add  it  to  9  cc.  of  the  liquefied  culture  medium  in  tube  No.  i ; 
i  cc.  of  tube  No.  i  to  tube  No.  2,  also  with  9  cc.  of  medium;  i  cc.  of  tube 
No.  2  to  tube  No.  3  (with  9  cc.  of  medium),  following  the  other  directions 
as  already  given.  Plate  out  as  already  explained,  and  watch  developments. 


BACTERIOLOGICAL    TECHNIC  87 

In  Petri  dish  No.  i  the  number  of  bacterial  growths  (colonies)  will  no  doubt 
be  so  great  as  to  make  counting  impossible.  Petri  dish  No.  2  may  contain 
360  colonies,  and  dish  No.  3  may  contain  not  nrore  than  40.  An  average 
is  obtained  by  repeating  the  test  (using  the  same  milk  sample)  a  number 
of  times.  In  the  above  milk  sample  the  average  may  be  42,000  microbes 
per  cc.  If  the  bacterial  content  is  high,  it  is  necessary  to  extend  the  dilu- 
tion four  and  even  five  times.  Usually  boiled  distilled  water  is  used 
with  which  to  make  desired  dilutions  (i-io,  i-ioo,  1-1,000,  1-10,000, 
etc). 

If  it  is  desired  to  determine  the  number  of  bacteria  per  gram  of  dry  soil, 
it  will  be  necessary  to  carefully  weigh  a  small  quantity  (i  gm.,  more  or  less) 
of  average  soil,  triturate  the  entire  sample  with  say,  100  cc.  of  sterile  dis- 
tilled water,  and  from  this  make  the  dilution  cultures  as  above  described, 
using  i  cc.  or  less  of  the  soil  triturate.  To  compute  the  number  of  bacteria 
per  gram  of  dry  soil,  it  will  now  be  necessary  to  determine  the  moisture  per- 
centage in  a  sample  of  soil  taken  from  the  same  place  as  the  sample  which 
was  used  in  making  the  triturate.  The  solution  is  simple.  We  will  suppose 
the  triturate  sample  weighed  0.856  gm.  and  the  number  of  bacteria  found 
was  3,000,000;  and  the  percentage  of  moisture  was  10.  From  these  data 
it  would  be  found  that  i  gm.  of  dry  soil  will  contain  3,855,011  microbes, 

The  above  is  sufficient  to  make  clear  how  one  might  proceed  to  deter- 
mine the  number  of  microbes  in  and  upon  old  pills,  tablets,  powders; 
on  one  ivory  vaccine  tip,  in  one  glycerinated  vaccine  tube,  in  i  cc.  of  bac- 
terial vaccine,  antitoxin,  syrup,  tincture,  fluidextract,  camphor  water, 
distilled  water,  sewage,  drinking  water,  etc.  Naturally,  great  caution  and 
care  must  be  observed  to  avoid  errors  and  faulty  conclusions.  In  fact, 
no  one  should  attempt  such  work  in  actual  practice  until  after  considerable 
preliminary  laboratory  experience. 

It  is  not  practicable  nor  is  it  necessary  to  give  fuller  information  regard- 
ing bacterial  cultures.  We  have  not  touched  upon  the  various  methods 
for  determining  whether  or  not  the  microbes  under  investigation  are 
essentially  aerobic  or  essentially  anaerobic;  the  manner  of  determining 
the  thermal  death-point;  relationship  of  rate  of  growth  to  temperature, 
etc.  We  have  said  nothing  of  the  use  of  indicators  added  to  culture  media, 
as  litmus,  rosolic  acid,  and  phenolphthale:n,  nor  have  we  explained  the 
special  use  of  special  culture  media  in  determining  the  nature  and  identity 
of  bacteria.  These  and  many  other  details  we  must  omit,  merely  stating 
that,  should  it  become  desirable  to  make  such  investigations,  the  necessary 
information  must  be  secured  elsewhere,  as  in  some  standard  laboratory 
guide  in  bacteriological  technic. 

The  following  outline  of  special  methods  will  serve  as  a  guide  in  making 
bacteriological  examinations  of  soils,  air,  pharmaceuticals,  liquids,  etc. 


88 


PHARMACEUTICAL   BACTERIOLOGY 


D.  Culturing  Soil  Bacteria. — Soil  is  a  mixture  of  dead  and  decayed 
organic  matter,  sand  and  living  organisms  and  their  spores.  Near  the 
surface  the  soil  contains  large  numbers  of  bacteria,  from  10,000  to  10,000,- 
ooo  per  gram,  and  more.  In  fact  the  fertility  of  the  soil  is  practically 
proportional  to  the  number  of  bacteria  present.  Most  species  of  soil 
bacteria  are  harmless  to  man  though  the  bacilli  of  tetanus  (lockjaw), 
of  typhoid  fever,  of  malignant  edema,  of  anthrax,  and  of  pus  formation 
may  be  present.  The  tetanus  germ  is  quite  common  in  garden  soils  and 
the  anthrax  germ  is  apt  to  occur  in  cattle  pens,  pastures  and  other  places 
frequented  by  cattle.  Other  soil  bacteria  are  decidedly  useful  as  will  be 
more  fully  explained  elsewhere. 


PIG.  37. — Graduated  fermentation  tube.     These  tubes  are  required  for  gas  determi- 
nation with  colon  bacillus  and  other  gas-forming  micro-organisms. 

Some  soil  bacteria  (the  nitrifiers)  do  not  grow  on  the  usual  media  while 
others  thrive  exceedingly  well  in  such  media.  Anaerobic  forms  must  be 
cultured  in  the  absence  of  air  or  oxygen. 

The  root  nodule  bacteria  of  the  leguminosae  can  be  grown  readily  on 
gelatin  or  agar .  The  tubercles  or  nodules  must  be  thoroughly  cleansed  and 
repeatedly  washed  in  boiled  distilled  water,  then  rinsed  for  ten  seconds  in  a 
i-iooo  corrosive  sublimate  solution,  and  finally  thoroughly  rinsed  (three 
minutes)  in  boiled  distilled  water.  Crush  several  of  the  sterilized  nodules 
in  a  sterile  watch  crystal,  by  means  of  a  sterile  glass  rod  and  from  this 
make  the  dilution  plate  cultures  and  set  aside  at  room  temperature.  Colo- 
nies of  small  motile  bacteria  (Rhizobium  mutabile)  will  appear  in  about  four 
days. 

To  test  the  soil  bacterially,  select  thoroughly  mixed  samples  and  plate 
out  as  already  suggested,  using  every  precaution  to  prevent  the  introduc- 
tion of  extraneous  germs.  Cultures  can  also  be  made  from  internal  plant 
tissues  by  following,  in  general,  the  directions  given  under  root  nodule 


BACTERIOLOGICAL   TECHNIC  89 

bacteria,  excepting  that  after  the  washing  and  rinsing,  the  root,  instead  of 
being  crushed,  is  cut  or  broken  across  and  the  inoculation  material  is 
taken  from  the  inner  tissue  by  means  of  a  platinum  needle  or  scalpel. 

E.  Bacteria  of  the  Air. — Air  currents  carry  the  germ-laden  dust  and  dirt 
particles.  The  number  and  kind  of  air  bacteria  depends  upon  environ- 
ment, climatic  conditions,  moisture,  sunlight,  etc.  The  air  currents  are 
the  main  factors  in  germ  dissemination.  Spores  and  dry  (though  not 
dead)  bacilli  may  be  carried  many  miles.  Air  microbes  are  derived  from 
the  soil  surface  and  from  the  objects  surrounded  by  the  air.  Bacteria 
are  exhaled  with  the  breath  (as  in  talking,  sneezing,  coughing)  and 
are  carried  and  distributed  from  and  by  animals,  plants  and  clothing. 

The  air  may  carry  organisms  derived  from  the  soil,  from  water  and 
from  other  substances  contaminated  by  organisms.  The  dirt  and  dust 
particles  wafted  about  by  air  currents  may  have  lodged  upon  them  the 
germs  of  tuberculosis,  the  pus  formers,  the  streptococcus  group,  rarely 
also  the  anthrax  bacillus,  the  tetanus  bacillus  and  the  bacillus  of  malignant 
oedema;  beside  the  spores  of  higher  fungi,  yeast  cells  and  even  the  larvae 
of  intestinal  parasites,  etc. 

Air  microbes  may  be  studied  by  exposing  a  Petri  dish  containing  steril- 
ized agar  or  gelatin,  for  two  minutes  or  longer.  The  number  of  colonies 
that  will  appear  will  depend  upon  the  locality,  season,  air  moisture,  etc. 
To  determine  the  number  of  microbes  in  a  given  volume  of  air  the  Sedg- 
wick-Tucker  aerobioscope  is  used,  though  similarly  constructed  apparatus 
may  be  made  by  any  fairly  skillful  student.  The  aerobioscope  consists  of  a 
glass  cylinder  as  shown  in  the  illustration.  The  open  ends  are  plugged 
with  cotton.  Granulated  sugar  is  loosely  packed  into  the  narrow  end 
and  all  is  then  sterilized  in  a  hot-air  sterilizer  (not  over  120°  C.).  Pass 
a  given  quantity  of  air  through  the  aerobioscope  by  attaching  an  aspirator 
bottle  to  the  narrow  end  and  allowing  a  given  volume  of  water  to  run  out 
of  the  bottle.  The  volume  of  air  drawn  through  equals  the  volume  of 
water  run  from  the  bottle.  Of  course  the  cotton  plug  is  removed  from  the 
larger  end  of  tube  while  the  water  is  running.  The  bacilli  and  spores  are 
caught  in  the  sugar,  while  the  air  passes  through.  Replace  cotton  plug 
and  shake  the  sugar  into  the  larger  end  of  tube.  Remove  cotton  plug 
again  and  pour  in  about  10  to  15  cc.  of  liquefied  (40°  C.,  not  hot)  gelatin. 
Roll  the  tube  held  horizontally.  The  gelatin  dissolves  the  sugar  and  mixes 
with  it.  Roll  on  ice  to  hasten  the  hardening  of  the  gelatin.  Set  aside  in 
incubator,  at  room  temperature  (20°  C.,  about).  The  number  of  colonies 
which  appear  indicates  approximately  the  number  of  microbes  in  the 
volume  of  air  aspirated.  Let  us  suppose  that  the  number  of  colonies  was 
125,  the  volume  of  air  aspirated  10  liters,  from  which  we  would  get  1250 
bacteria  per  cubic  meter  of  air. 


90  PHARMACEUTICAL  BACTERIOLOGY 

F.  Bacteria  of  Liquid  Substances. — The  bacteria  of  water,  milk,  tinc- 
tures, fluid  extracts,  aquae,  aerated  waters,  mineral  waters,  distilled  water, 
broth,  and  liquids  generally,  can  be  studied  quantitatively  in  a  compara- 
tively simple  manner.  By  means  of  a  sterile  i  cc.  graduated  pipette, 
run  o.i  cc.  to  0.5  cc.  of  the  liquid  into  the  center  of  a  sterilized  petri  dish, 
pour  upon  this  enough  (about  10  cc.)  melted  (sterile)  agar  or  gelatin  and 
mix  by  tilting  the  dish  slightly  from  side  to  side.  Set  aside  for  the  medium 
to  harden  and  incubate  at  the  room  temperature,  or  at  37°  C.,  as  may  be 
required.  This  method  is  satisfactory  if  the  number  of  bacilli  present  is 
comparatively  small.  If  very  abundant,  dilutions  must  be  made  in  the 
manner  already  described. 

The  following  general  suggestions  should  be  observed  in  making  bac- 
teriological determinations  of  liquids : 


FIG.  38. — Aerobioscope  after  Sedgwiek- Tucker,  plugged  with  cotton.     The  larger  end 
in  which  the  culturing  is  done  is  ruled  to  facilitate  the  counting  of  colonies. 

a.  Containers  for  samples  (other  than  the  original  containers)  must  be 
sterile  and  closed  with  sterile  corks  or  cotton  plugs.     If  the  samples  are  to 
be  carried  any  distance  they  should  be  packed  in  ice.     In  no  case  is  it  wise 
to  keep  a  sample  longer  than  forty-eight  hours  before  culturing  it.     If  the 
sample  is  to  be  examined  within  two  or  three  hours  after  collecting  it, 
placing  on  ice  is  not  absolutely  necessary. 

b.  Every  sample  should  be  thoroughly  mixed  before  making  cultures. 
Shake  well,  about  twenty  times.     This  is  very  important. 

c.  All  glassware,  pipettes,  etc.,  must  be  thoroughly  sterilized  by  wash- 
ing, rinsing,  wiping,  hot  air  or  steam  sterilization,  etc. 

d.  The  methods  of  making  dilutions,  the  amounts  to  be  planted  or 
tubed,  the  culture  media  to  be  used,  etc.,  etc.,  cannot  be  given  in  detail  in 
a  work  of  this  kind.     All  will  depend  upon  the  kind  of  analysis  to  be 
made,  the  results  to  be  attained,  etc.     The  special  methods  to  be  em- 
ployed in  special  cases  must  be  looked  up  in  suitable  text-books  and 
carefully  followed.     The  following  general  suggestions  are  in  order  at  this 
time. 

e.  Thus  far  there  are  no  standards  for  the  bacteriological  testing  of 
Pharmaceuticals.     Tinctures   and   fluidextracts   should  show  only   few 
colonies  per  cc.,  not  over  30  to  60.     Sera  should  show  none.     Well  pre- 
pared and  properly  ripened  small-pox  vaccine  should  show  only  a  few 
colonies,  from  20  to  500,  per  ivory  point  or  per  tube. 


BACTERIOLOGICAL    TECHNIC  91 

f.  The  colon  bacillus  should  not  be  present  in  specified  volumes  of 
drinking  water,  of  milk  and  pharmaceuticals.     If  present  in  such  vol- 
umes, it  indicates  excessive  sewage  or  other  objectionable  contamination. 
The  colon  bacillus  is  motile  in  young  broth  cultures,  forms  no  spores,  is 
gas-  (dextrose  broth  cultures  in  fermentation  tube)  and  indol-forming, 
reduces  nitrates  to  nitrites,  does  not  liquefy  gelatin  and  is  not  stained 
by  Gram's  method. 

g.  Syrups  of  all  kinds,  unless  very  carefully  prepared  and  carefully  kept 
to  prevent  fermentation,  are  apt  to  show  numerous  bacteria,  yeasts  and 
molds.    Any  syrup  showing  signs  of  yeast  fermentation  (gas  bubbles, 
vinous  odor)  or  moldiness,  it  not  fit  for  use  and  should  be  rejected.     The 
attempt  to  render  it  usable  by  boiling,  is  unsatisfactory,  furthermore  the 
changes  produced  by  the  organisms  are  always  objectionable  and  cannot  be 
rectified  by  heating  or  by  other  methods  of  sterilization. 

h.  Recent  investigations  have  shown  that  many  of  the  marketed 
(bottled)  mineral  waters  contain  numerous  bacteria,  from  10,000  to 
300,000,000  and  more  per  cc.  In  some  cases  colon  bacilli  have  been  found. 
These  findings  prove  that  in  many  instances  the  methods  of  bottling  must 
be  careless  or  otherwise  unsatisfactory.  Undoubtedly  the  contamina- 
tion is  in  some  instances  due  to  reused  and  inadequately  cleaned  and 
sterilized  containers  and  in  other  instances  to  impure  and  inadequately 
sterilized  mineral  water.  A  popular  opinion  prevails  that  the  chemicals  in 
the  mineral  waters  are  sufficiently  germicidal  to  destroy  bacteria  but 
this  is  not  the  case. 

G.  Bacteria  in  Canned  Fruits. — The  work  recently  demanded  by  the 
pure  food  laws  (federal  and  state)  has  shown  that  such  food  substances  as 
canned  fruits  of  all  kinds,  including  jams,  jellies,  preserves,  catsups, 
tomato  pastes,  etc.,  are  frequently  highly  contaminated  with  yeast  cells, 
molds  and  their  spores,  and  other  higher  fungi,  and  bacteria.  It  is, 
however  evident  that  the  food  products  named  may  be  kept  quite  free 
from  such  contamination  as  may  be  seen  from  the  examination  of  canned 
food  products  prepared  by  the  careful  housewife.  That  manufacturers 
may  approximate  the  home  condition  is  demonstrated  by  the  fact  that 
factory  products  are  found  on  the  market,  which  are  quite  free  from 
contamination. 

Since  wholesome  ripe  fruit  contains  yeast  cells,  bacteria  and  mold  in 
very  small  numbers  only4,  and  since  most  of  these  organisms  are  removed  in 
the  various  steps  of  the  processing,  as  washing,  peeling,  steaming,  etc.,  it  is 
evident  that  the  finished  factory  product  should,  like  the  home:made 
product,  contain  these  organisms  in  negligibly  small  numbers  only,  pro- 
vided, of  course,  that  wholesome  fruit  is  used.  However,  most  of  the 
factory  samples  thus  far  examined  have  shown  numerous  dead  yeast  cells, 


92  PHARMACEUTICAL  BACTERIOLOGY 

mould  spores,  mould  hyphae,  and  bacteria,  indicating  the  use  of  fruit,  fruit 
pulp,  fruit  juices,  fruit  refuse,  etc.,  which  was  decomposed  or  undergoing 
fermentation  or  decomposition  prior  to  or  at  the  time  of  manufacture. 
The  organisms  named  prevail  in  varying  amounts  in  different  products. 
Yeast  organisms  are  apt  to  predominate  in  jellies,  fruit  juices  and  fruit 


FIG.  39. — Thoma-Zeiss  hemacytometer.  Complete  equipment  for  blood  counting. 
This  is  very  convenient  for  making  bacterial  counts  in  catsups,  jams,  jellies  and  other 
vegetable  foods  and  also  in  animal  food  substances. 

pulp;  bacteria  in  catsups   and   pastes;  and  molds   in  certain  fruits  as 
strawberries,  blackberries  and  raspberries. 

The  presence  of  numerous  dead  yeast  cells  (1,000,000  to  50,000,000  per 
cc.)  is  evidence  that  the  material  was  undergoing  alcoholic  fermentation 


FIG.  40. — Zappert  ruling  of  the  Thoma-Zeiss  hemacytometer.  This  form  of 
ruling  is  especially  convenient  for  making  bacterial  counts  and  counts  of  fat  globules 
in  milk. — (Carl  Zeiss.} 

just  prior  to  or  at  the  time  of  manufacture.  Tomato  pastes  have  been 
found  on  the  market  showing  over  35,000,000,000  bacteria  per  cc.  besides 
numerous  yeast  cells  and  considerable  mold.  The  bacterial  content  of 
catsups  is  apt  to  run  high,  from  50,000,000  to  500,000,000  and  more  per  cc. 
Not  including  the  vinegar  bacteria,  which  are  introduced  into  catsups  and 


BACTERIOLOGICAL   TECHNIC  93 

pastes,  such  high  bacterial  content  is  generally  due  to  bacterial  develop- 
ment during  or  after  manufacture.  The  presence  of  mold  organisms  and 
their  spores  (other  than  Penicillium)  indicates  the  use  of  mold-infested 
fruit.  Penicillium,  which  is  entirely  saprophytic  in  habit,  may  develop 
after  manufacture,  particularly  on  the  surface  of  inadequately  sterilized 
fruit  products  in  containers  not  entirely  rilled. 

" Swelling"  of  cans  containing  fruit  products  is  generally  due  to  yeast 
development,  though  it  may  also  be  due  to  bacterial  activity,  and  indicates 
inadequate  sterilization  of  either  the  container  or  of  the  fruit  or  both. 
Examination  will  show  the  presence  of  living  yeast  cells,  or  bacteria,  per- 
haps air  bubbles,  and  the  characteristic  vinous  odor  of  yeast  maybe  noted. 

Based  upon  such  conditions  as  can  be  made  to  prevail  in  carefully  oper- 
ated factories,  the  following  may  be  given  as  the  limits  of  the  number  of 
organisms  permissible  in  the  fruit  products  under  discussion. 

a.  Yeast  cells,  either  living  or  dead,  not  to  exceed  1,000,000  per  cc. 

b.  Mold  spores  not  to  exceed  1,000,000  per  cc. 

c.  Hyphal  clusters  and  hyphal  fragments  not  to  exceed  10,000  per  cc.; 
or  not  over  25  per  cent,  of  separate  and  distinct  fields  of  view  under  the 
compound  microscope  should  show  hyphal  clusters  or  hyphal  fragments. 

d.  Bacteria  (either  living  or  dead  but  not  including  vinegar  bacteria  in 
products  to  which  vinegar  is  added  )not  to  exceed  25,000,000  per  cc. 

The  above  figures  apply  only  to  fruit  products  supposedly  made  from 
comparatively  fresh  fruits  and  fresh  fruit  juices.  The  yeast,  bacterial  and 
spore  counts  are  made  with  a  Thoma-Zeiss  hemacy tometer  (Turck  ruling) 
using  a  No.  5  (%  in.)  objective  with  No.  2  (i  in.)  ocular. 

H.  Quantitative  and  Qualitative  Bacteriological  Testing. — The  following 
will  serve  as  a  general  outline  of  bacteriological  analyses  which  may  be  made 
in. food  and  drug  laboratories.  The  substances  which  require  such  bac- 
teriological examination  include  catsups,  tomato  pastes,  vinegars,  water 
supplies,  mineral  waters,  milk,  ice  creams,  any  and  all  substances  which 
are  suspected  to  be  sewage  contaminated,  etc.,  etc. 

The  sequence  of  processes  here  given  bear  a  progressive  relationship. 
Whether  process  II  is  carried  out  will  depend  upon  the  findings  under  I 
and  whether  III  shall  be  undertaken  will  depend  upon  the  findings  under  II. 
The  essential  facts  to  be  ascertained  are  whether  or  not  there  is  possible 
sewage  contamination  as  indicated  by  the  presence  of  the  colon  bacillus, 
sewage  streptococci  and  possibly  the  typhoid  bacillus.  The  typhoid 
agglutinating  tests  are  apt  to  prove  unsatisfactory.  In  most  instances 
this  test  will  be  unnecessary  as  the  presence  of  the  colon  bacillus  is  evidence 
that  the  food,  drug  or  drink  is  excessively  contaminated  with  sewage 
and  is  hence  unfit  for  human  use. 

I.  Direct  Count. — For  this  purpose  the  Thoma-Zeiss  hemacytometer 


94  PHARMACEUTICAL  BACTERIOLOGY 

with  Turck  ruling1  is  used  (No.  2  ocular  with  %  in.  objective)  which  can  be 
secured  from  any  bacteriological  supply  house.  The  instructions  for 
using  it  can  be  obtained  from  the  dealer,  though  the  measuring  values 
indicated  on  the  hemacytometer  are  sufficient  to  indicate  the  manner  of 
making  the  counts.  The  rulings  generally  used  for  bacterial  countings  are 
Jioo  sq-  mm-  X  Mo  mm-  deep,  making  an  area  of  Mooo  cu.  mm.,  or  re- 
duced to  decimal  fractions,  0.04  sq.  mm.  X  o.i  mm.  deep  =  0.004  cu.  mm. 
We  will  suppose  that  the  average  of  20  counts  shows  5  bacilli,  then  i  cu. 
mm.  would  contain  20,000  bacilli  or  20,000,000  in  i  cc. 

The  direct  count  is,  in  many  instances,  very  unsatisfactory  for  several 
reasons.  Particles  other  than  micro-organisms  may  be  mistaken  for  bacilli 
or  cocci  and,  furthermore,  it  cannot  be  known  for  a  certainty  that  the  organ- 
isms are  dead  or  alive.  If  they  are  present  in  great  abundance  (10,000,000 
to  100,000,000  and  more  per  cc.),  ordinary  smear  preparations  may  be 
stained,  using  methyl  blue  or  Hoffmann's  violet.  Dead  bacilli,  that  is 
those  which  have  been  dead  for  some  time,  do  not  take  the  stain  well, 
due  to  the  fact  that  the  cellplasm  is  disintegrated. 

Tomato  pastes,  anchovy  pastes,  catsups,  some  mineral  waters  and 
imilar  preparations,  may  contain  bacteria  in  such  numbers  that  dilutions 
are  desirable  or  necessary  to  make  counting  possible.  Dilutions  of 
i-io,  i-ioo  will,  as  a  rule,  be  sufficient.  Weigh  or  measure  one  part 
(i  gm.  or  i  cc.)  of  the  substance,  add  it  to  9  or  99  parts  filtered  distilled 
water  and  mix  thoroughly  by  shaking. 

If  the  direct  count  shows  bacilli  in  great  numbers  or  if  for  any  reason 
sewage  contamination  is  suspected,  and  also  to.determine  the  number  of 
living  bacilli  and  spores  suspected,  proceed  as  follows: 

II.  Plate  Culture  Counts. — Make  one  set  of  plate  cultures,  using  lactose 
litmus  agar,2  and  incubate  at  20°  C.  Make  a  second  set  of  plate  cultures, 
also  upon  lactose  litmus  agar,  and  incubate  at  38°  C.  The  usual  dilution 
methods  are  followed  when  necessary,  using  preferably  o.i  cc.  quantities 
for  the  plates.  This  temperature  differential  test  is  considered  of  great 
importance.  Colon  bacilli  and  other  micro-organisms,  whose  natural 
habitat  is  the  intestinal  canal,  will  develop  actively  at  the  higher  tempera- 
ture (38°  C.),  whereas  the  usual  air,  soil  and  water  bacteria  develop  best 
at  the  lower  temperature  (20°  C.).  If  the  high  temperature  colonies  ap- 
proximate the  low  temperature  colonies,  sewage  contamination  may  be 
suspected.  If  in  addition  many  of  the  high  temperature  lactose  litmus 
agar  colonies  show  pink  or  light  vermilion,  the  sewage  contamination  is 
practically  proven.  The  colon  bacillus,  as  well  as  sewage  streptococci, 

1  To  render  the  ruled  lines  visible  rub  a  very  soft  pencil  over  the  ruled  area. 
2  Add  i  per  cent,  of  lactose  to  the  usual  agar  medium  and-enough  tincture  of  litmus 
to  give  it  a  lilac  tinge. 


BACTERIOLOGICAL    TECHNIC  95 

give  pink  colonies,  the  latter  being  the  brighter,  more  vermilion  in  colora- 
tion, due  to  the  formation  of  acid  (in  the  fermenting  lactose).  Examine 
the  pink  colonies  under  the  microscope.  The  colon  microbe  is  rod-shaped, 
rather  thick,  non-sporing,  and  shows  motility  in  recent  broth  cultures, 
whereas  the  streptococci  are  smaller  and  are  not  rod-shaped.  High  tem- 
perature colonies  as  compared  with  low  temperature  colonies  should  not 
exceed  i  :ioo  or  1 125.  If  the  proportion  is  1 14  or  less,  sewage  contamina- 
tion is  very  likely.  After  36  hours  the  pink  colonies  may  turn  blue,  due 
to  the  development  of  ammonia  and  amines. 

Naturally  the  high  temperature  colonies  must  be  studied  within 
twenty-four  to  thirty  hours  whereas  the  low  temperature  cultures  require 
much  more  time,  two  to  four  days. 

If  the  temperature  and  color  differential  tests  indicate  sewage  con- 
tamination, then  the  following  additional  tests  should  be  carried  out. 

III.  Indol  Reaction  and  Gas  Formula. — The  indol  reaction  has  already 
been  explained.  The  gas  formula  is  determined  as  follows:  To  sets  of 
four  graduated  fermentation  tubes  containing  glucose  bouillon  and  lac- 
tose bouillon,  add  o.i,  0.2,  0.5,  and  10  cc.  of  the  suspected  liquid  and 
incubate  at  38°  for  48  hours.  If  gas  formation  is  observed  the  presence 
of  colon  bacilli  may  be  suspected.  If  the  o.i  cc.  tubes  show  gas  forma- 
tion then  the  presence  of  colon  bacilli  may  be  assumed.  Fill  the  bulb 
of  a  tube,  showing  gas  formation,  with  a  2-per  cent,  solution  of  sodic 
hydrate,  hold  thumb  tightly  over  the  opening  and  mix  contents  by  tilting 
back  and  forth  carefully.  The  portion  of  gas  absorbed  is  COz  whereas 
the  unabsorbed  portion  is  supposedly  hydrogen.  The  colon  bacillus 
shows  a  gas  formation  of  %  hydrogen.  Of  course  the  total  volume 
of  gas  is  recorded  before  the  sodic  hydrate  is  added. 

The  gas  formula  with  a  positive  indol  reaction  is  practically  conclusive 
as  far  as  the  presence  of  the  colon  bacillus  is  concerned.  Add  to  this  the 
other  tests  and  we  have  conclusive  evidence  of  sewage  contamination. 

The  colon  bacillus,  the  bacilli  of  the  hog  cholera  group  and  others, 
have  the  power  of  reducing  neutral  red;  producing  a  greenish-yellow 
fluorescence.  For  this  reaction  use  glucose  bouillon  to  which  has  been 
added  i  per  cent,  of  a  0.5  per  cent,  solution  of  neutral  red.  In  examining 
milk,  the  pus  cell  and  leucocyte  count  is  considered  important;  centrifu- 
galize  10  cc.  of  milk  for  five  minutes,  pour  off  supernatant  milk  and 
mix  sediment  with  0.5  cc.  normal  salt  solution  and  make  counts  of  pus 
cells  and  leucocytes  per  cc.  from  the  amount  (0.5  cc.) .  Abundant  pus  cells 
and  leucocytes  indicate  abscess  or  other  pathological  condition  of  milk 
ducts  or  glands.  This  test  is,  however,  of  little  significance  excepting  in 
the  hands  of  authorities  on  diseases  of  cows.  It  is  stated  that  as  many  as 
100,000  leucocytes  per  cc.  may  occur  in  apparently  healthy  animals . 


96  PHARMACEUTICAL  BACTERIOLOGY 

Gelatin-liquefying  organisms  may  be  looked  upon  with  suspicion 
when  found  in  milk,  water  and  other  liquid-food  substances  intended  for 
human  consumption,  as  has  already  been  explained. 

It  should  be  borne  in  mind  that  the  colon  bacillus  is  one  of  a  group  of 
some  fifteen  or  more  species  and  varieties  of  closely  related  micro-organ- 
isms which  resemble  each  other  in  the  following  particulars: 

1.  Do  not  form  spores.  . 

2.  Do  not  liquefy  gelatin. 

3.  Produce  acid  in  milk  and  cause  milk  coagulation. 

4.  Produce  acid  and  gas  in  glucose  and  lactose  media. 

5.  Produce  acid  and  gas  in  bile-salt-glucose  broth. 

6.  Grow  well  at  temperatures  ranging  from  38°  to  42°  C. 

In  differentiating  the  colon  bacillus,  remember  that  this  organism  is 
rod-shaped  (2  to  3^  long  by  0.5  to  0.6/4  wide),  is  motile,  produces  indol, 
gives  rise  to  pink  colonies  on  lactose  (or  glucose)  litmus  agar  and  reduces 
neutral  red  glucose  (or  lactose)  agar  with  a  greenish-yellow  fluorescence. 

It  should  also  be  remembered  that  sewage  is  a  highly  complex  substance 
and  contains  micro-organisms  in  great  variety  and  in  great  abundance. 
Among  the  organisms  present  are  species  of  Spirillum,  Vibrio,  Proteus  and 
Beggiatoa  in  addition  to  the  bacilli  and  streptococci  already  mentioned. 
The  typhoid  bacillus  does  not  thrive  well  in  sewage.  The  number  of  bac- 
teria present  in  crude  or  ordinary  sewage  (domestic,  city,  hospital,  mixed, 
etc.)  ranges  from  1,000,000  to  100,000,000  and  more  per  cc.  The  work 
of  these  organisms  is  to  break  down  and  render  soluble  and  assimilable 
(for  plants)  the  organic  matter  composing  the  sewage,  thus  assisting  the 
work  of  rotting  bacteria  generally. 

The  following  is  a  tabulation  of  the  bacteriological  testing  that  should 
be  made  of  foods  (including  pastes,  catsups,  milk,  ice  creams,  water  sup- 
plies, mineral  waters,  alcoholic  beverages,  etc.)  that  may  show  an  excess 
of  bacterial  growth  or  which  may  be  sewage  contaminated: 

BACTERIOLOGICAL  EXAMINATION 

I.  Direct  Count. 

1.  Bacilli  per  cc 

2.  Cocci,  per  cc 

II.  Plate  and  Tube  Cultures.     (Lactose-litmus-agar.) 

1.  Temperature  differential  test. 

a.  (20°  C.)  Colonies  per  cc 

b.  (38°  C.)  Colonies  per  cc 

2.  Color  differential  test. 

a.  Pink  colonies  per  cc 

c.  Not  pink  colonies  per  cc 


BACTERIOLOGICAL   TECHNIC  97 

3.  Colorless  gelatin  liquefying  colonies  per  cc. 

4.  Neutral  red  reduction,  +  or  — . 

5.  Indol  reaction,  +  or  — . 

6.  Gram  stain  behavior,  +  or  — . 

7.  Gas  (hydrogen)  formula. 

III.  Agglutinating  tests  for  Typhoid  Germs. 
i 

8.  Staining  Bacteria 

Staining  consists  of  the  infiltration  of  the  cell-substance  with  solutions 
of  various  coloring  materials  obtained  for  the  most  part  from  the  group  of 
coal-tar  derivatives  known  as  the  aniline  dyes.  As  is  generally  known, 
different  cells  and  different  portions  of  one  and  the  same  cell  react  differ- 
ently with  the  various  dyes  used.  This  peculiar  behavior  brings  out 
contrasts  in  appearances  which  aid  very  materially  in  determining  the 
morphological  characters.  The  prime  object,  therefore,  in  using  stains  is 
to  aid  in  the  study  of  cell  morphology.  Different  bacteria  react  differ- 
ently with  the  several  stains  used.  Some  species  take  certain  stains  very 
readily,  while  they  are  quite  indifferent  to  other  stains.  The  vegetative 
cell  stains  much  more  readily  than  do  the  spores.  In  fact,  spores  are 
stained  with  great  difficulty;  however,  after  they  are  once  thoroughly 
stained  they  hold  the  color  persistently. 

The  dyes  which  may  be  used  in  bacteriologic  work  are  of  many  kinds, 
differing  as  to  color  and  as  to  staining  powers  with  different  cells,  cell-con- 
tents, and  cell-parts.  They  are  usually  classified  as  acid  or  basic.  Eosin, 
acid  fuchsin,  and  picric  acid  are  acid  stains,  and  are  said  to  be  diffuse 
in  their  effects,  having  no  special  affinity  for  any  special  cell  structure, 
fuchsin,  methylene  blue,  and  gentian  violet  are  basic,  and  appear  to  have 
special  attraction  for  bacteria  and  for  plasmic  and  nuclear  substances  of 
cells  generally,  for  which  reasons  they  are  most  generally  employed  as 
bacterial  stains.  Fuchsin  is,  in  fact,  about  the  only  efficient  stain  for 
endospores,  while  gentian  violet  and  methylene  blue  are  excellent  stains 
for  the  bacterial  cell-wall. 

It  is  known  that  certain  substances  possess  the  property  of  preparing 
the  bacterial  cells  in  such  a  way  as  to  induce  them  to  take  up  the  dye 
more  readily,  thus  intensifying  the  stain,  as  aniline  oil  and  carbolic  acid. 
Such  substances  are  called  mordants,  and  may  be  used  separately  or  added 
directly  to  the  stain  itself. 

Certain  liquids  or  solutions  remove  the  stain  from  the  bacterial  cell 
more  or  less  readily,  as  water  and  alcohol,  but  more  particularly  solutions 
of  acids.  Such  substances  are  quite  generally  employed  for  removing  any 
excess  of  stain  from  the  bacterial  cell  or  from  the  matrix  in  which  the  bac- 


98  PHARMACEUTICAL  BACTERIOLOGY 

teria  are  fixed  or  embedded.  Acidulated  (with  HC1)  alcohol  is  most 
commonly  employed.  Ordinarily,  rinsing  in  a  small  stream  of  water  is 
sufficient.  Some  bacteria  resist  the  decolorizing  process  with  acids  more 
strongly  than  others,  and  are  said  to  be  acid  fast  or  acid  proof,  as,  for 
example,  the  bacilli  of  leprosy  and  of  tuberculosis,  while  the  great  majority 
of  species  give  up  the  stain  very  readily.  It  is  a  fa  ct  that  one  and  the  same 
species  of  microbe  reacts  variably  with  one  and  the  same  stain,  depending 
upon  a  variety  of  causes.  Moderate  heat  hastens  and  intensifies  the 
staining. 

For  ordinary  purposes  a  single  stain  only  is  used,  but  sometimes  struc- 
tural differences  are  more  clearly  shown  by  what  is  known  as  double  or  con- 
trast staining.  Take,  for  example,  a  spore-bearing  microbe,  as  that  of 
anthrax.  The  spores  may  be  stained  by  means  of  carbol  f  uchsin ;  the  en- 
tire cell,  excepting  the  spore,  can  be  completely  decolorized  in  acidulated 
alcohol,  and  then  methylene  blue  or  gentian  violet  applied  as  the  contrast 
stain.  We  then  have  a  blue  cell-wall  with  a  red  spore.  However,  the 
beginner  is  apt  to  be  disappointed  in  his  attempts  at  double  staining. 

The  pharmacist  will  have  comparatively  little  to  do  as  far  as  the  actual 
staining  of  bacteria  is  concerned.  He  should,  however,  be  able  to  pre- 
pare the  more  important  stains,  mordants,  and  other  solutions  which 
may  be  required  by  the  city  or  health  board  bacteriologist  or  the  phy- 
sician, and  we  shall  therefore  give  the  more  commonly  employed 
preparations. 

A.  Stock  Solutions. — Make  saturated  solutions  of  the  basic  dyes  (f  uch- 
sin, gentian  violet,  and  methylene  blue)  in  95  per  cent,  alcohol.     Keep 
these  in  glass-stoppered  bottles  in  a  cool,  dark  place,  ready  for  use  in  pre- 
paring the  stains.     The  stock  solutions  should  in  all  instances  be  filcered 
before  using.     Secure  the  dyes  from  reliable  dealers  and  in  small  quantities . 
Do  not  make  up  large  quantities  of  stock  solutions  or  stains  proper,  as 
they  gradually  deteriorate,  particularly  if  exposed  to  light. 

B.  Mordants. — The  principal  substances  used  are  aniline,  carbolic  acid, 
tannic  acid,  glacial  acetic  acid,  ferrous  sulphate,  sodium  hydroxide  solu- 
tion, chromic  acid,  and  a  few  others.     Those  in  general  use  are  the  two 
first  named.     The  others  have  a  more  limited  use  in  special  cases. 

i.  Aniline  Water 

Aniline,  2  cc. 

Distilled  Water,  98  cc. 

Shake  frequently,  and  finally  filter  several  times  through  filter  paper. 
It  should  be  perfectly  clear.  This  preparation  deteriorates  rapidly. 
Make  up  small  amounts  and  keep  in  a  dark  place.  It  becomes  worthless, 
even  when  observing  all  precautions,  in  a  few  weeks. 


BACTERIOLOGICAL   TECHNIC  gy 

2.  Carbolic  Acid  Solution 

Carbolic  Acid,  20  cc. 

Distilled  Water,  100  cc. 

Filter.     This  mordant  is  rarely  used  by  itself. 

C.  Stains. — We  give  here  the  more  important  stains,  approximately  in 
the  order  of  preferred  use. 

i.  Loeffler's  Methylene  Blue 

Stock  Solution  (saturated)  Methylene  Blue,  30  cc. 

1:10,000  Sol.  KHO  in  Dist.  Water,  100  cc. 

Mix,  shake  filter.  This  stain  is  much  used  as  a  general  bacterial  stain 
and  in  the  examination  of  blood,  pus,  etc. 

2.  Aniline  Gentian-Violet 

Aniline  Water,  75  cc. 

Stock  Solution  Gentian-Violet,  25  cc. 

Mix,  shake,  filter.     This  is  an  excellent  bacterial  stain. 

3.  Carbol-Fuchsin 

Stock  Solution  of  Basic  Fuchsin,  10  cc. 

5  per  cent.  Sol.  Carbolic  Acid,  100  cc. 

Mix,  shake,  filter.  This  is  one  of  the  most  useful  stains  with  the 
so-called  acid-proof  microbes.  It  is  also  a  spore  stain,  and  is  the  most 
commonly  employed  stain  used  in  contrast  or  double  staining.  It  is  a 
comparatively  slow  stain,  but  is  quite  permanent. 

4.  Gram's  Stain 

Gram's  stain  is  used  for  diagnostic  purposes,  and  is  perhaps  the  best 
known  stain  in  the  entire  field  of  bacteriological  technic.  Its  value  de- 
pends upon  the  fact  that  certain  microbes,  when  stained  and  afterward 
treated  with  a  solution  of  iodine  and  washed  in  alcohol,  give  up  the  stain. 
Such  microbes  are  known  as  Gram-negative,  whereas  thqse  which  do  not 
give  up  the  stain  are  said  to  be  Gram-positive. 

The  method  of  using  this  stain  is  somewhat  complicated,  requires  care, 
and,  with  a  beginner,  often  yields  disappointing  results.  Keeping  in  mind 
the  following  will  minimize  the  disappointments : 

a.  Long-continued  (one  year  or  more)  subcultures  frequently  lose  the 
Gram-stain  behavior. 

b.  Old  cultures,  that  is,  those  which  have  been  growing  in  the  same 
medium  for  several  days  or  more,  as  a  rule  do  not  stain  characteristically. 
With  such  cultures  the  results  are  often  neither  negative  nor  positive,  just 
enough  to  be  confusing  and  perplexing. 

c.  The  solutions  used  must  be  fresh.     The  gentian-aniline  solution,  as 
well  as  the  iodine  solution,  deteriorates  quite  rapidly. 


100  PHARMACEUTICAL  BACTERIOLOGY 

d.  Do  not  overstain,  and  do  not  decolorize  too  long.  Stop  decolorizing 
as  soon  as  no  more  violet  color  comes  away. 

In  the  Gram  method  two  solutions  are  used,  namely: 

1.  Aniline  gentian- violet,  and 

2.  Gram's  iodine  solution. 

Iodine,  i  gm. 

Potassium  Iodide,  2  gm. 

Distilled  Water,  300  cc. 

The  method,  briefly  outlined,  is  as  follows: 

a.  Spread  the  bacteria  evenly  and  thinly  over  the  cover-glass  (the 
usual  smear  preparation).     Stain  with  the  aniline  gentian- violet  for  from 
two  to  five  minutes.     Warming  will  hasten  and  intensify  the  staining. 
Wash  in  water  to  remove  excess  of  stain. 

b.  Drop  on  the  iodine  solution  and  allow  it  to  act  for  about  one  min- 
ute or  until  the  preparation  assumes  a  coffee-brown  color.     It  may  be 
desirable  to  apply  the  iodine  a  second  time. 

c.  Wash  off  the  excess  of  iodine  in  water  and  then  decolorize  by  drop- 
ping on  95  per  cent,  alcohol.     Tip  the  slide  and  allow  alcohol  to  run  over 
the  preparation;  continue  until  the  violet  color  ceases  to  stream  away. 

d.  Finally  rinse  in  water  and  examine  in  water.     If  desired,  dry  and 
mount  permanently  in  Canada  balsam  or  some  other  suitable  mounting 
medium. 

e.  A  contrast  stain,  such  as  eosin,  fuchsin,  safranin,  or  Bismarck 
brown,  may  be  used,  following  (c). 

Keeping  in  mind  the  difficulties  already  referred  to  in  using  the  Gram 
method,  and  the  additional  possible  source  of  error  due  to  the  fact  that 
one  and  the  same  microbe  will  stain  but  feebly  at  one  time  and  very 
intensely  at  another  time,  we  now  name  the  principal  organisms  which 
are  Gram-positive  or  Gram-negative. 

Bacteria  and  other  Organisms  Stained  by  the  Gram  Method 

Staphylococcus  pyogenes  aureus. 

Staphylococcus  pyogenes  albus. 

Streptococcus  pyogenes. 

Micrococcus  tetragenus. 

Micrococcus  lanceolatus. 

Bacillus  diphtheriae. 

Bacillus  tuberculosis. 

Bacillus  of  anthrax. 

Bacillus  of  tetanus. 

Bacillus  of  leprosy. 

Bacillus  aerogenes  capsulatus. 

Oidium  albicans. 

Actinomyces  (of  Actinomycosis). 


BACTERIOLOGICAL   TECHNIC  IOI 

Bacteria  not  Stained  by  the  Gram  Method 

Diplococcus  of  meningitis  (intracellular) . 

Diplococcus  of  gonorrhea. 

Micrococcus  melitensis. 

Bacillus  of  chancroids  (Ducrey's). 

Bacillus  of  dysentery  (Shiga's). 

Bacillus  of  typhoid  fever. 

Bacillus  of  bubonic  plague. 

Bacillus  of  influenza. 

Bacillus  coli  communis. 

Bacillus  pyocyaneus. 

Bacillus  of  Friedlander. 

Bacillus  proteus. 

Bacillus  mallei  (glanders). 

Spirillum  of  Asiatic  cholera. 

Spirillum  of  relapsing  fever. 

5.  Pappenheim's  Stain 

Sat.  Aqueous  Sol.  Methyl  Green,  50  cc. 

Sat.  Aqueous  Sol.  Pyronine,  15  cc. 

Mix  and  filter.  This  is  much  used  for  staining  bacteria  in  pus  and 
other  pathological  secretions.  The  bacteria  are  stained  a  bright  red,  while 
the  cell  nuclei  are  blue  to  purple. 

6.  Smith's  Stain 

Stock  Sol.  Basic  Fuchsin,  10  ee. 
Methyl  Alcohol, 

Formaldehyde,  each     10  cc. 

Distilled  Water,  to  make  100  cc. 

Mix  and- filter.  Let  stand  for  twenty-four  hours  before  using.  Renew 
in  three  weeks.  The  stain  is  much  used  to  distinguish  between  bacteria 
and  nuclear  substances.  Allow  the  stain  to  act  for  from  two  to  ten 
minutes. 

7.  Flagella  Staining 

Care  is  necessary  in  staining  flagellae.  Numerous  methods  have  been 
recommended,  but  Pitfield's  method,  as  modified  by  Muir,  is  perhaps  the 
best  and  at  the  same  time  comparatively  simple.  The  following  solutions 
are  required: 

a.  Mordant 

Tannic  Acid  (10  per  cent.  Aq.  Sol.),  10  cc. 

Sat.  Aq.  Sol.  Mercuric  Chlor.,  5  cc. 

Sat.  Aq.  Sol.  Alum,  5  cc. 

Carbol-Fuchsin,  5  cc. 

Mix,  shake,  filter  or  centrifuge.  This  soluton  does  not  keep  longer 
than  one  week. 


IO2  PHARMACEUTICAL  BACTERIOLOGY 

b.  Stain 

Sat.  Aq.  Sol.  Alum,  10  cc. 

Stock  Sol.  Gentian- Violet,  2  cc. 

Mix,  filter.     Carbol-fuchsin  may  be  used  instead  of  gentian- violet. 
This  stain  will  not  keep  longer  than  a  few  days. 
The  method  is  as  follows : 

1.  Drop  on  mordant.    Leave  for  one  minute,  with  gentle  heat. 

2.  Rinse  in  water  for  two  minutes. 

3.  Dry  carefully  at  slight  warmth. 

4.  Stain  for  one  minute  with  gentle  heat. 

5.  Wash,  dry,  and  mount  in  Canada  balsam. 

In  making  the  cover-glass  preparation,  take  a  loopful  from  a  young 
^aqueous  subculture  of  some  motile  bacillus  and  touch  it  on  the  carefully 
cleaned  cover  and  allow  the  drop  to  spread  by  rotating  and  tilting  the  cover. 
Do  not  use  the  loop  more  than  is  necessary.  Flagellae  are  very  delicate  and 
easily  destroyed.  Dry  very  carefully,  and  do  not  pass  through  flame  more 
than  three  times. 

8.  Spore  Staining 

As  already  stated,  spores  (endospores)  of  microbes  stain  with  great 
difficulty,  for  which  reason  a  contrast  is  effected  negatively;  that  is,  the 
rest  of  the  cell  is  quickly  stained,  leaving  the  unstained,  highly  refractive 
spore  to  appear  like  a  bit  of  glass  within  the  colored  frame.  This  is  in 
many  ways  the  most  satisfactory  way  of  demonstrating  the  presence  of 
spores.  The  spores  may,  however,  be  stained  by  the  usual  acid-fast  or 
acid-proof  methods,  care  being  observed  in  decolorizing.  Stain  with 
hot  carbol-fuchsin  for  a  few  minutes,  wash,  and  decolorize  quickly  with 
3  per  cent,  hydrochloric  acid  in  95  per  cent,  alcohol,  and  then  use  a  con- 
trast stain,  as  gentian- violet  or  methylene  blue.  The  red  spores  will  then 
appear  in  the  violet  or  blue  frame. 

9.  Capsule  Staining 

The  gelatinous  capsule  of  microbes  is  also  stained  with  great  difficulty, 
and  requires  special  methods  and  experience  to  yield  anything  like  satis- 
•factory  results.  The  methods  of  Welch  and  Hiss  are  quite  satisfactory. 
The  capsule  is,  however,  generally  visible  without  any  staining  because 
of  the  light  contrast  that  naturally  exists.  Certain  substances,  as  glacial 
acetic  acid  (Welch  method),  cause  the  capsule  to  enlarge  and  take  up  the 
stain  more  readily.  Certain  staining  methods  bring  out  the  capsule  of 
certain  microbes,  as,  for  example,  the  Gram  method  as  applied  to  pneu- 
monia sputum. 


BACTERIOLOGICAL   TECHNIC  103 

The  Muir  method  is  perhaps  the  best  for  capsule  staining.  It  is  as 
follows : 

1.  Stain  in  carbol-fuchsin  for  one-half  minute,  with  gentle  heat. 

2.  Wash  lightly  in  alcohol  (95  per  cent.)- 

3.  Wash  well  in  water. 

4.  Flood  with  mordant  of 

Sat.  Aq.  Sol.  Mercuric  Chlor.,  2  cc. 

Tannic  Acid  (20  per  cent.  Aq.  Sol.),  2  cc. 

Sat.  Aq.  Sol.  Potassium  Alum,  5  cc. 

5.  Wash  in  water. 

6.  Wash  in  95  per  cent,  alcohol,  one  minute. 

7.  Wash  in  water. 

8.  Stain  with  methylene  blue  for  one-half  minute. 

9.  Decolorize  somewhat  and  let  dry. 

10.  Clear  in  xylene,  and  mount  in  Canada  balsam. 

There  are  numerous  other  special  stains  and  special  staining  methods, 
which  need  not  be  mentioned  here.  Should  the  pharmacist  be  called  upon 
to  prepare  any  of  these,  he  will  find  full  particulars  in  any  standard  work 
on  medical  bacteriology. 

9.  Studying  Bacteria 

The  complete  study  of  any  one  species  of  microbe  with  a  view  to  deter- 
mining its  identity  is  a  long  and  tedious  process.  It  involves  a  study  of  the 
organism  in  its  natural  element  and  in  artificial  culture  media,  and  its 
behavior  in  animal  inoculation  tests,  etc.  Special  apparatus,  experimental 
animals  (as  rats,  mice,  guinea-pigs,  dogs,  etc.),  and  technical  experience 
and  skill  are  necessary.  Just  what  kind  of  observations  are  involved  in 
such  study  is  indicated  in  the  complete  method  as  outlined  by  the  Society 
of  American  Bacteriologists  (Jan.,  1908),  which  is  hereby  submitted  for 
the  benefit  of  those  who  may  wish  to  acquaint  themselves  with  such  details. 
The  glossary  of  terms  should  be  carefully  considered  first  of  all.  The  deci- 
mal system  for  indicating  groups  relationships  of  microbes  (Table  I)  is  most 
unique  and  is  very  convenient  for  active  workers.  Those  interested  will 
find  the  desired  explanations  of  the  methods  and  reagents  mentioned,  in  any 
of  the  larger  works  on  medical  bacteriology  and  on  bacteriological  tech- 
nology. It  is  not  at  all  likely  that  the  pharmacist  will  ever  have  occasion 
to  make  use  of  the  special  methods  cited.  He  should  nevertheless  acquaint 
himself  with  them  sufficiently  to  comprehend  their  application  in  the  study 
of  pathogenic  bacteria. 

Our  bacteria  nomenclature  is  in  some  confusion,  and  unless  the  methods 
of  naming  bacteria  are  corrected,  the  confusion  is  certain  to  become  much 
greater.  The  trouble  lies  in  the  failure  to  define  group  or  generic  delimita- 


104  PHARMACEUTICAL  BACTERIOLOGY 

tions.  The  present  generic  terms,  "bacillus  "  and  "  micrococcus, "  include 
too  many  species.  We  have  a  confusing  and  almost  incomprehensible 
array  of  synonyms,  of  which  those  applied  to  Rhizobium  mutabile  may  serve 
as  an  example.  The  different  names  that  have  been  given  to  this  organism 
may  be  arranged  as  follows : 

Pasteuraceae,  Laurent. 
Bacteria,  Woronin,  1866. 
Bakteroiden,  Brunchorst  and  Frank,  1885. 
Microsymbiont,  Atkinson,  1893. 
Spores  or  gemmules,  Ward  and  Ericksson. 
Bacillus  radicicola,  Beyerinck,  1888. 
Cladochylrium  leguminosarum,  Vuellemin. 
Phytomyxa  leguminosarum,  Schroeter. 
Schinzia  leguminosarum,  Woronin. 
Rhizobium  leguminosarum,  Frank,  1890 
Rhizobium  Frankii,  (in  part)  Schneider,  1892. 
Rhizobium  mutabile,  Schneider,  1902. 
Pseudomonas  radicicola,  Moore,  1905. 
Rhizobium  leguminosarum.     The  Com.  1917. 

The  above  synonomy  is  also  interesting  because  it  indicates  a  most 
remarkable  difference  of  opinion  regarding  the  nature  and  identity  of  this 
root-nodule  organism.  Further,  as  the  result  of  the  wholly  inadequate 
group  delimitations  we  have  such  name-monstrosities  as  Granulobacillus 
saccharobutyricus  mobilis  nonliquifaciens,  and  M icrococcus  acidi  paralactici 
liquifaciens  Halensi.  Reform  in  nomenclature  is  very  desirable,  and  it 
must  come  through  a  careful  definition  of  generic  groups  based  on 
physiological  characters,  rather  than  upon  largely  morphological 
characters,  as  is  done  now. 

It  is  advised  that  the  pharmacist  refrain  from  experimenting  with 
pathogenic  organisms,  excepting  in  so  far  as  he  may  act  in  cooperation  with 
practicing  physician  or  health  officers.  When  experimenting  with  patho- 
genic organisms  the  greatest  caution  is  necessary  to  guard  against  autoin- 
oculation  and  the  spreading  of  disease.  It  should  be  made  a  rule  to  treat 
every  microbe  studied  as  though  it  were  virulently  pathogenic,  capable 
of  spreading  an  epidemic.  Never  expose  a  colony  (plate  culture,  tube 
culture,  etc.)  in  such  a  way  as  to  peimit  the  escape  of  the  organisms  into 
the  air.  Pour  a  disinfecting  solution  (5  per  cent,  carbolic  acid)  into  cul- 
tures that  are  to  be  discontinued  and  then  boil  container  and  all,  for  thirty 
minutes,  before  washing  and  cleaning  the  glassware.  Never  forget  to  steri- 
lize the  platinum  needle  before  and  after  making  an  inoculation  or  a  culture 
transfer. 


BACTERIOLOGICAL   TECHNIC  105 

DESCRIPTIVE    CHART— SOCIETY    OF    AMERICAN     BACTERIOLOGISTS1 

GLOSSARY  OF  TERMS 

Agar  Hanging  Block,  a  small  block  of  nutrient  agar  cut  from  a  poured  plate,  and  placed, 
on  a  cover-glass,  the  surface  next  the  glass  having  been  first  touched  with  a  loop  from 
a  young  fluid  culture  or  with  a  dilution  from  the  same.  It  is  examined  upside  down, 
the  same  as  a  hanging  rock. 

Ameboid,  assuming  various  shapes  like  an  ameba. 

Amorphous,  without  visible  differentiation  in  structure. 

Arborescent,  a  branched,  tree-like  growth. 

Beaded,  in  stab  or  stroke,  disjointed  or  semi-confluent  colonies  along  the  line  of  inocula- 
tion. 

Brief,  a  few  days,  a  week. 

Brittle,  growth  dry,  friable  under  the  platinum  needle. 

Bullate,  growth  rising  in  convex  prominences,  like  a  blistered  surface. 

Butyrous,  growth  of  a  butter-like  consistency. 

Chains,  short  chains,  composed  of  2  to  8  elements.  Long  chains,  composed  of  more  than 
8  elements. 

Ciliate,  having  fine  hair-like  extensions,  like  cilia. 

Cloudy,  said  of  fluid  cultures  which  do  not  contain  pseudozooglea. 

Coagulation,  the  separation  of  casein  from  whey  in  milk.  This  may  take  place  quickly 
or  slowly,  and  as  the  result  either  of  the  formation  of  an  acid  or  of  a  lab  ferment. 

Contoured,  an  irregular,  smoothly,  undulating  surface,  like  that  of  a  relief  map. 

Convex,  surface  the  segment  of  a  circle,  but  flattened. 

Coprophyl,  dung  bacteria. 

Coriaceous,  growth  tough,  leathery,  not  yielding  to  the  platinum  needle. 

Crateriform,  round,  depressed,  due  to  the  liquefaction  of  the  medium. 

Cretaceous,  growth  opaque  and  white,  chalky. 

Curled,  composed  of  parallel  chains  in  wavy  strands,  as  in  anthrax  colonies. 

Diastasic  Qction,  same  as  Diastatic,  conversion  of  starch  into  water-soluble  substances  by 
diastase. 

Echinulate,  in  agar  stroke  a  growth  along  the  line  of  inoculation,  with  toothed  or 
pointed  margins;  in  sag  cultures  growth  beset  with  pointed  outgrowths. 

Effuse,  growth  thin,  veily,  unusually  spreading. 

Entire,  smooth,  having  a  margin  destitute  of  teeth  or  notches. 

Erase,  border  irregularly  toothed. 

Filamentous,  growth  composed  of  long,  irregularly  placed  or  interwoven  filaments. 

Filiform,  in  stroke  or  stab  cultures  a  uniform  growth  along  line  of  inoculation. 

Fimbriate,  border  fringed  with  slender  processes,  larger  than  filaments. 

Floccose,  growth  composed  of  short  curved  chains,  variously  oriented. 

Flocculent,  said  of  fluids  which  contain  pseudozooglea,  i.e.,  small  adherent  masses  of 
bacteria  of  various  shapes  and  floating  in  the  culture  fluid. 

Fluorescent,  having  one  color  by  transmitted  light  and  another  by  reflected  light. 

Gram's  Stain,  a  method  of  differential  bleaching  after  gentian- violet,  methyl- violet,  etc. 
The  -f  mark  is  to  be  given  only  when  the  bacteria  are  deep  blue  or  remain  blue  after 
counters! aining  with  Bismarck  brown. 

Grumose,  clotted. 

1  Prepared  by  F.  D.  Chester,  F.  P.  Gorham,  Erwin  F.  Smith,  Committee  on 
Methods  of  Identification  of  Bacterial  Species.  Endorsed  by  the  Society  for  general  use 
at  the  annual  meeting,  January,  1908. 


106  PHARMACEUTICAL  BACTERIOLOGY 

Infundibuliform,  form  of  a  funnel  or  inverted  cone. 

Iridescent,  like  mother-of-pearl.     The  effect  of  very  thin  films. 

Lacerate,  having  the  margin  cut  into  irregular  segments  as  if  torn. 

Lobate,  border  deeply  undulate,  producing  lobes  (see  Undulate}. 

Long,  many  weeks  or  months. 

Maximum  Temperature,  temperature  above  which  growth  does  not  take  place. 

Medium,  several  weeks. 

Membranous,  growth  thin,  coherent,  like  a  membrane. 

Minimum  Temperature,  temperature  below  which  growth  does  not  take  place. 

Mycelioid,  colonies  having  the  radial  ely  filamentous  appearance  of  mould  colonies. 

Napiform,  liquefaction  with  the  form  of  a  turnip. 

Nitrogen  Requirements,  the  necessary  nitrogenous  food.  This  is  determined  by  adding 
to  nitrogen-free  media  the  nitrogen  compound  to  be  tested. 

Opalescent,  resembling  the  color  of  an  opal. 

Optimum  Temperature,  temperature  at  which  growth  is  most  rapid. 

Pellicle,  in  fluid  bacterial  growth  either  forming  a  continuous  or  an  interrupted  sheet 
over  the  fluid. 

Peptonized,  said  of  curds  dissolved  by  trypsin. 

Persistent,  many  weeks,  or  months. 

Pseudozooglece,  clumps  of  bacteria,  not  dissolving  readily  in  water,  arising  from  imper- 
fect separation,  or  more  or  less  fusion  of  the  components,  but  not  having  the  degree 
of  compactness  and  gelatinization  seen  in  zooglea. 

Puhinate,  in  the  form  of  a  cushion,  decidely  convex. 

Punctiform,  very  minute  colonies,  at  the  limit  of  natural  vision. 

Rapid,  developing  in  twenty-four  to  fqrty-eight  hours. 

Raised,  growth  thick,  with  abrupt  or  terraced  edges. 

Repand,  wrinkled. 

Rhizoid,  growth  of  an  irregular  branched  or  root-like  character,  as  in  B.  mycoides. 

Ring,  same  as  Rim,  growth  at  the  upper  margin  of  a  liquid  culture,  adhering  more  or  less 
closely  to  the  glass. 

Saccate,  liquefaction  the  shape  of  an  elongated  sac,  tubular,  cylindrical. 

Scum,  floating  islands  of  bacteria,  an  interrupted  pellicle  or  bacterial  membrane. 

Slow,  requiring  five  or  six  days  or  more  for  development. 

Short,  applied  to  time,  a  few  days,  a  week. 

Sporangia,  cells  containing  endospores. 

Spreading,  growth  extending  much  beyond  the  line  of  inoculation,  i.e.,  several  milli- 
meters or  more. 

Stratiform,  liquefying  to  the  walls  of  the  tube  at  the  top  and  then  proceeding  downward 
horizontally. 

Thermal  Death- point,  the  degree  of  heat  required  to  kill  young  fluid  cultures  of  an  organ- 
ism exposed  for  ten  minutes  (in  thin-walled  test-tubes  of  a  diameter  not  exceeding 
20  mm.)  in  the  thermal  water-bath.  The  water  must  be  kept  agitated  so  that 
the  temperature  shall  be  uniform  during  the  exposure. 

Transient,  a  few  days. 

Turbid,  cloudy  with  flocculent  particles;  cloudy  flocculence. 

Umbonate,  having  a  button-like,  raised  center. 

Undulate,  border  wavy  with  shallow  sinuses. 

Verrucose,  growth  wart-like,  with  wart-like  prominences. 

Vermiform-contoured,  growth  like  a  mass  of  worms,  or  intestinal  coils. 

Villous,  growth  beset  with  hair-like  extensions. 


BACTERIOLOGICAL   TECHNIC  1 07 

Viscid,  growth  follows  the  needle  when  touched  and  withdrawn,  sediment  on  shaking 

rises  as  a  coherent  swirl. 
Zooglea,  firm  gelatinous  masses  of  bacteria,  one  of  the  most  typical  examples  of  which  is 

the  Streptococcus  mesenter aides  of  sugar  vats  (Leuconostoc  mesenterioides),  the  hac-_ 

terial  chains  being  surrounded  by  an  enormously  thickened  firm  covering  inside  of 

which  there  may  be  one  or  many  groups  of  the  bacteria. 

NOTES 

(1)  For  decimal  system  of  group  numbers  see  Table  i.     This  will  be  found  useful  as 
a  quick  method  of  showing  close  relationships  inside  the  genus,  but  is  not  a  sufficient 
characterization  of  any  organism. 

(2)  The  morphological  characters  shall  be  determined  and  described  from  growths 
obtained  upon  at  least  one  solid  medium  (nutrient  agar)  and  in  at  least  one  liquid 
medium  (nutrient  broth).     Growths  at  37°  C.  shall  be  in  general  not  older  than  twenty- 
four  to  forty-eight  hours,  and  growths  at  20°  C.  not  older  than  forty-eight  to  seventy-two 
hours.     To  secure  uniformity  in  cultures,  in  all  cases  preliminary  cultivation  shall  be 
practised  as  described  in  the  revised  Report  of  the  Committee  on  Standard  Methods  of 
the  Laboratory  Section  of  the  American  Public  Health  Association,  1905. 

(3)  The  observation  of  cultural  and  bio-chemical  features  shall  cover  a  period  of  at 
least  fifteen  days  and  frequently  longer,  and  shall  be  made  according  to  the  revised 
Standard  Methods  above  referred  to.     All  media  shall  be  made  according  to  the  same 
Standard  Methods. 

(4)  Gelatin  stab  cultures  shall  be  held  for  six  weeks  to  determine  liquefaction. 

(5)  Ammonia  and  indol  tests  shall  be  made  at  end  of  tenth  day,  nitrate  tests  at  end 
of  fifth  day. 

N 

(6)  Titrate  with  —  NaOH,  using  phenolphthalein  as  an  indicator:  make  titrations  at 

same  time  from  blank.     The  difference  gives  the  amount  of  acid  produced. 

The  titrations  should  be  done  after  boiling  to  drive  of  any  COa  present  in  the  culture. 

(7)  Generic  nomenclature  shall  begin  with  the  year  1872  (Cohen's  first  important 
paper). 

Species  nomenclature  shall  begin  with  the  year  1880  (Koch's  discovery  of  the  poured 
plate  method  for  the  separation  of  organisms). 

(8)  Chromogenesis  shall  be  recorded  in  standard  color  terms. 

TABLE  i 

A  NUMERICAL  SYSTEM  OF  RECORDING  THE  SALIENT  CHARACTERS  OF  AN   ORGANISM 

(GROUP  NUMBER.) 

100 Endospores  produced. 

200 Endospores  not  produced. 

10 Aerobic  (Strict). 

20 Facultative  anaerobic. 

30 Anaerobic  (Strict). 

i Gelatin  liquefied. 

2 Gelatin  not  liquefied. 

o.i Acid  and  gas  from  dextrose. 

0.2 Acid  without  gas  from  dextrose. 

0.3 No  acid  from  dextrose. 


IO8  PHARMACEUTICAL  BACTERIOLOGY 

0.4 No  growth  with  dextrose. 

.01 Acid  and  gas  from  lactose. 

.02 Acid  without  gas  from  lactose. 

.03 No  acid  from  lactose. 

.04 No  growth  with  lactose. 

.001 Acid  and  gas  from  saccharose'. 

.  002 Acid  without  gas  from  saccharose. 

.003 No  acid  from  saccharose. 

.004 No  growths  with  saccharose. 

.0001 Nitrates  reduced  with  evolution  of  gas. 

.0002 Nitrates  not  reduced. 

.0003 Nitrates  reduced  without  gas  formation. 

.  ooooi Fluorescent. 

.00002 Violet  chromogens. 

.  00003 Blue  chromogens. 

.00004 Green  chromogens. 

.  00005 Yellow  chromogens. 

.00006 Orange  chromogens. 

.  00007 Red  chromogens. 

.00008 Brown  chromogens. 

.00009 Pink  chromogens. 

.00000 Non-chromogenic. 

.000001 Diastasic  action  on  potato  starch,  strong. 

.000002 Diastasic  action  on  potato  starch,  feeble. 

.  000003 Diastasic  action  on  potato  starch,  absent. 

.  ooooooi Acid  and  gas  from  glycerin. 

.0000002 Acid  without  gas  from  glycerin. 

.  0000003 No  acid  from  glycerin. 

.0000004 No  growth  with  glycerin. 

The  genus  according  to  the  system  of  Migula  is  given  its  proper  symbol  which  pre- 
cedes the  number  thus:  (7) 

BACILLUS  COLI  (Esch.)  Mig.  becomes B.  222.111102 

BACILLUS  ALCALIGENES  Petr.  becomes B.  212 .333102 

PSEUDOMONAS    CAMPESTRis    (Pam.)    Sm.    be- 
comes  Ps.  211.333151 

BACTERIUM  SUICIDA  Mig.  becomes Bact.  222 .  232103 


DETAILED  FEATURES 

NOTE. — Underscore  required  terms.     Observe  notes  and  glossary  of  terms. 

I.       MORPHOLOGY  (2) 

i.       Vegetative  Cells,  Medium  used temp ....  age days 

Form,  round,  short  rods,  long  rods,  short  chains,  long  chains,  filaments,  commas,  short 
spirals,  long  spirals,  clostridium,  cuneate,  clavate,  curved. 

Limits  of  Size 

Size  of  Majority 

Ends,  rounded,  truncate,  concave. 


Agar 
Hanging-block 


BACTERIOLOGICAL   TECHNIC  1 09 

Orientation  (grouping) 

Chains  (No.  of  elements) 

Short  chains,  long  chains. 


Orientation  of  chains,  parallel,  irregular. 

2.  Sporangia,  medium  used temp age days 

Form,  elliptical,  short  rods,  spindled,  clavate,  drum-sticks. 
Limits  of  Size Size  of  Majority 

f  Orientation  (grouping) 

Agar  <  Chains  (No.  of  elements) 

Hanging-block    [  Orientation  of  Chains,  parallel,  irregular. 
Location  of  Endospores,  central,  polar. 

3.  Endospores. 

Form,  round,  elliptical,  elongated. 

Limits  of  Size 

Size  of  Majority 

'  Wall,  thick,  thin. 

Sporangium  wall,  adherent,  not  adherent. 
Germination,  equatorial,  oblique,  polar,  bipolar,  by  stretching. 

4.  Flagella,  No Attachment,  polar,  bipolar,  peritrichiate.     How  Stained 

5.  Capsules,  present  on 

6.  Zooglea,  Pseudozooglea. 

7.  Involution  Forms,  on in days  at °C. 

8.  Staining  Reactions. 

1:10  watery  fuchsin,  gentian-violet,  carbol-fuchsin.     Loeffler's  alkaline  methylene 

blue. 
Special  Stains 

Gram Glycogen 

Fat Acid-fast 

Neisser 

II.     CULTURAL  FEATURES  (3) 

1.  A  gar  Stroke. 

Growth,  invisible,  scanty,  moderate,  abundant. 

Form  of  growth,  filiform,  echinulate,  beaded,  spreading,  plumose,  arborescent,  rhizoid. 

Elevation  of  growth,  flat,  effuse,  raised,  convex. 

Luster,  glistening,  dull,  cretaceous. 

Topography,  smooth,  contoured,  rugose,  verrucose. 

Optical  Characters,  opaque,  translucent,  opalescent,  iridescent. 

Chromogenesis  (8) 

Odor,  absent,  decided,  resembling 

Consistency,  slimy,  buytrous,  viscid,  membranous,  coriaceous,  brittle. 
Medium  grayed,  browned,  reddened,  blued,  greened. 

2.  Potato. 

Growth,  scanty,  moderate,  abundant,  transient,  peristent. 

Form  of  growth,  filiform,  echinulate,  beaded,  spreading,  plumose,  arborescent,  rhizoid. 

Elevation  of/growth,  flat,  effuse,  raised,  convex. 

Luster,  glistening,  dull,  cretaceous. 

Topography ]  smoot h,  contoured,  rugose,  verrucose. 

Chromogenesis  (8) Pigment  in  water  insoluble,  soluble;  other 

solvents . . 


IIO  PHARMACEUTICAL   BACTERIOLOGY 

Odor,  absent,  decided,  resembling 

Consistency,  slimy,  butyrous,  viscid,  membranous,  coriaceous,  brittle. 
Medium  grayed,  browned,  reddened,  blued,  greened. 

3.  Loeffler's  Blood-serum. 

Stroke  invisible,  scanty,  moderate,  abundant.     Form  of  growth,  filiform,  echinulate, 

beaded,  spreading,  plumose,  arborescent,  rhizoid. 
Elevation  of  growth,  flat,  effuse,  raised,  convex. 
Luster,  glistening,  dull,  cretaceous. 
Topography,  smooth,  contoured,  rugose,  verrucose. 

Chromogenesis  (8) 

Medium  grayed,  browned,  reddened,  blued,  greened. 

Liquefaction  begins  in d,  complete  in d. 

4.  A  gar  Stab. 

Growth,  uniform,  best  at  top,  best  at  bottom;  surface  growth  scanty,  abundant;  restricted 

widespread. 
Line  of  puncture,  filiform,  beaded,  papillate,  villous,  plumose,  arborescent:  liquefaction. 

5.  Gelatin  Stab. 

Growth,  uniform,  best  at  top,  best  at  bottom. 

Line  of  puncture,  filiform,  beaded,  papillate;  villous,  plumose,  arborescent. 

Liquefaction,  crateriform,  napiform,  infundibuliform,  saccate,  stratiform;  begins  in 

d,  complete  in d. 

Medium  fluorescent,  browned 

6.  Nutrient  Broth. 

Surface  growth,  ring,  pellicle,  occulent,  membranous,  none. 

Clouding,  slight,  moderate,  strong;  transient,  persistent;  none;  fluid  turbid. 

Odor,  absent,  decided,  resembling. 

Sediment,  compact,  occulent,  granulay,  flaky,  viscid  on  agitation,  abundant,  scant. 

7.  Milk. 

Clearing  without  coagulation. 
Coagulation  prompt,  delayed,  absent. 

Extrusion  of  whey  begins  in days. 

Coagulum  slowly  peptonized,  rapidly  peptonized. 

Peptonization  begins  on d,  complete  on d. 

Reaction,  id ,  2d ,  4d ,  lod ,  2od 

Consistency,  slimy,  viscid,  unchanged. 
Medium  browned,  reddened,  blued,  greened. 
Lab  ferment,  present,  absent. 

8.  Litmus  Milk. 

Acid,  alkaline,  acid  then  alkaline,  no  change. 
Prompt  reduction,  no  reduction,  partial  slow  reduction. 

9.  Gelatin  Colonies. 
Growth  slow,  rapid. 

Form,  punctiform,  round,  irregular,  ameboid,  mycelioid,  filamentous,  rhizioid. 
Elevation,  flat,  effuse,  raised,  convex,  pulvinate,  crateriform  (liquefying}. 
Edge,  entire,  undulate,  lobate,  erase,  lacerate,  fimbriate,  filamentous,  floccose,  curled. 
Liquefaction,  cup,  saucer,  spreading. 
10.  A  gar  Colonies. 

Growth  slow,  rapid  (temperature ). 

Form,  punctiform,  round,  irregular,  ameboid,  mycieloid,  filamentous,  rhizoid 


BACTERIOLOGICAL   TECHNIC 


III 


Surface  smooth,  rough,  concentrically  ringed,  radiate,  striate 
Elevation,  flat,  effuse,  raised,  convex,  puhinate,  umbonate. 
Edge,  entire,  undulate,  lobate,  erose,  lacerate,  fimbriate,  floccose,  curled. 
Internal  structure,   amorphous,  finely-,  coarsely-granular,   grumose,    filamentous, 
floccose,  curled. 

11.  Starch  Jelly. 

Growth,  scanty,  copious. 

Diastasic  action,  absent,  feeble,  profound. 

Medium  stained 

12.  Silicate  Jelly  (Fermi's  Solution). 
Growth  copious,  scanty,  absent. 
Medium  stained 

13.  Cohn's  Solution. 

Growth  copious,  scanty,  absent. 
Medium  fluorescent,  non-fluorescent. 

14.  Uschinsky's  Solution. 
Growth  copious,  scanty,  absent. 
Fluid  viscid,  not  viscid. 

15.  Sodium  Chloride  in  Bouillon. 
Per  cent,  inhibiting  growth. 

1 6.  Growth  in  Bouillon  over  Chloroform,  unrestrained,  feeble,  absent. 

17.  Nitrogen.     Obtained   from   peptone,   asparagin,   glycocoll,   urea,    ammonia  salts, 

nitrogen. 

18.  Best  media  for  long-continued  growth 

19.  Quick  tests  for  differential  purposes 


III.  PHYSICAL  AND  BIOCHEMICAL  FEATURES. 


i.  Fermentation  tubes  con- 
taining peptone-water  or 
sugar-free    bouillon    and 

Dextrose 

Saccharose 

Lactose 

Maltose 

Glycerin 

Mannit 

Gas  production,  in  per  cent. 

(*_\ 

\coJ 

Growth  in  closed  arm 

Amount  of  acid  produced  id. 

Amount  of  acid  produced  2d. 

Amount  of  acid  produced  3d. 

- 

2.  Ammonia  production,  feeble,  moderate,  strong,  absent,  masked  by  acids. 

3.  Nitrates  in  nitrate  broth. 
Reduced,  not  reduced. 


112 


PHARMACEUTICAL  BACTERIOLOGY 


Presence  of  nitrites ammonia 

Presence  of  nitrates free  nitrogen 

4.  Indol  production,  feeble,  moderate,  strong. 

5.  Toleration  of  Acids:  Great,  medium,  slight. 
Acids  tested. 

6.  Toleration  of  NaOH:  Great,  medium,  slight. 

7.  Optimum  reaction  for  growth  in  bouillon,  stated  in  terms  of  Fuller's  scale 

8.  Vitality  on  culture  media:  Brief,  moderate,  long. 

9.  Temperature  relations: 

Thermal  death-point  (10  minutes'  exposure  in  nutrient  broth  when  this  is  adapted 
to  growth  of  organism) C. 

Optimum  temperature  for  growth C.:  or  best  growth  at  15°  C.,  20°  C.,  25° 

C.,  30°  C.,  37°  C.,  40°  C.,  50°  C.,  60°  C. 

Maximum  temperature  for  growth C. 

Minimum  temperature  for  growth C. 

10.  Killed  readily  by  drying:  resistant  to  drying. 

11.  Per  cent,  killed  by  freezing  (salt  and  crushed  ice  or  liquid  air) 

12.  Sunlight:  Exposure  on  ice  in  thinly  sown  agar  plates,  one-half  plate  covered  (times 

15  minutes),  sensitive,  not  sensitive. 
Per  cent,  killed 

13.  Acids  produced 

14.  Alkalies  produced 

15.  Alcohols 

16.  Ferments:  Pepsin,  trypsin,  diastase,  invertase,  pectase,  cytase,  tyrosinase,  oxidase,  per- 

oxidase,  lipase,  catalase,  glucase,  galactase,  lab,  etc 

17.  Crystals  formed 

1 8.  Effect  of  germicides: 


Substance               Method  used 

Minutes 

Temper- 
ature 

Killing 
quantity 

Amt.  required  to 
restrain  growth 

IV.  PATHOGENICITY. 

1.  Pathogenic  to  animals. 

Insects,  crustaceans,  fishes,  reptiles,  birds,  mice,  rats,  guinea-pigs,  rabbits,  dogs, 
cats,  sheep,  goats,  cattle,  horses,  monkeys,  man. 

2.  Pathogenic  to  Plants: 


3.  Toxins,  soluble,  endotoxins. 

4.  Non-toxin  forming. 

5.  Immunity  bactericidal. 

6.  Immunity  non-bactericidal. 

7.  Loss  of  virulence  on  culture-media:  Prompt,  gradual,  not  observed  in months. 


BACTERIOLOGICAL    TECHNIC 


BRIEF  CHARACTERIZATION 

Mark  +  or  O,  and  when  two  terms  occur  on  a  line  erase  the  one  which  does  not 
apply  unless  both  apply. 


Diameter  over  iju 

Chains,  filaments 

Endospores 

Capsules 

Zooglea,  Pseudozooglea 

O      Motile 

Involution  forms 

Gram's  Stain 

f  Cloudy,  turbid 

Broth 

Pellicle 

I  Sediment 

Shining 

.Dull 

jg         gar  I  Wrinkled 

Chromogenic 

Round 

Proteus-like 

^  i       Gel.  Plate        }  Rhizoid 

'  •  "•    '  Filamentous 

Curled 

Surface-growth 

Gel.  Stab.        \ ...     ,. 
J  Needle-growth 

Moderate,  absent 

Abundant 

Potato 

Discolored 

Starch  destroyed 

Grows  at  37°  C 

Grows  in  Cohn's  Sol I 

Grows  in  Uschinsky's  Sol j 

[Gelatin  (4) 

I  Blood^serum _ 

Liquefaction    <  „ 

Casein 

I  Agar,  mannan 

[Acid  curd 

g  c*  Milk  \  Rennet  curd 

[  Casein  peptonized 

Indol  (5) 

Hydrogen  suphide 

Ammonia  (5) 

Nitrates  reduced  (5) 

Fluorescent 

Luminous. . , 


114  PHARMACEUTICAL  BACTERIOLOGY 


Animal  pathogen,  epizoon  

J5 

Plant  pathogen,  epiphyte  

2 

Soil  

H 

Milk  

P 

PP  ' 

Fresh  water  

2 

Salt  water  

ft 

Sewage  

S 

Iron  bacterium  

Sulphur  bacterium  

A.  Counting  Plate  Colonies. — If  the  colonies  in  a  Petri  dish  culture  are 
few,  not  exceeding  fifty  to  one  hundred,  they  may  readily  be  counted  in 
full.     If  the  colonies  are  quite  numerous,  the  counting  may  be  made  easier 
by  marking  off  (by  means  of  a  grease  pencil  or  chalk)  the  bottom  of  the 
plate  into  two  right  angled  cross-lines  (quarter  sectors)  and  these  again 
into  equal  parts  (%  sectors) .     Or  one  of  the  recommended  special  counting 
plates  may  be  used.     Either  the  square  or  circular  plate  will  answer  the 
purpose  (see  figures).    When  colonies  are  very  numerous  (200  and  more) 
in  a  plate  culture  and  quite  uniformly  distributed,  it  is  not  necessary  to 
count  them  all.     Count  the  colonies  in  a  number  of  squares  or  sector  areas 
(square  centimeters)  and  multiply  the  average  of  twenty  counts  by  the 
number  of  squares  representing  the  entire  surface  area  of  the  culture 
plate.    As  a  rule  the  counting  should  be  complete,  however. 

From  the  plate  counts  it  is  possible,  by  simple  mathematics,  to  deter- 
mine the  number  of  microbes  in  the  dilution  cultures  of  water,  milk,  tinc- 
tures, fluidextracts,  as  has  already  been  explained. 

Studying  Plate  Colonies. — The  plate  colonies  should  be  studied  macro- 
scopically  and  also  with  the  aid  of  a  pocket  lens  and  under  the  low  power  of 
the  compound  microscope.  Place  the  dish  on  the  stage  of  the  microscope 
and  focus  upon  the  colonies  carefully  by  means  of  the  coarse  adjustment. 
Note  color,  outline  and  other  characteristics  of  the  colonies,  etc.,  as  already 
set  forth  under  tube  cultures  and  in  the  official  methods  of  the  Society  of 
Bacteriologists. 

B.  Making  Tube-cultures  (Subcultures). — Inoculate  test-tubes  (con- 
taining gelatin,  agar  or  other  media)  with  such  colonies  as  it  is  desired  to 
study  further.    This  is  done  as  follows:  Hold  the  test-tube  to  be  inocu- 
lated in  left  hand.     Take  up  the  platinum  needle  (straight  or  loop)  in  the 
right  hand  and  pass  the  entire  needle  and  glass  rod  (excepting  the  end 
held)  through  the  flame  of  a  Bunsen  burner  several  times;  heat  the  needle 
to  a  glowing  red  for  a  few  seconds  and  then  allow  it  to  cool  a  few  seconds. 
Lift  the  cover  of  the  Petri  dish  high  enough  to  pass  the  needle  under,  touch 
end  of  the  platinum  needle  (straight  or  loop)  on  colony  desired;  let  the 


BACTERIOLOGICAL    TECHNIC 


dish  cover  drop  into  place  again;  remove  the  cotton  plug  from  test-tube 
by  grasping  it  between  two  fingers  (back  of  fingers  toward  the  test-tube); 
make  the  inoculation  (deep  stab,  shallow  stab,  or  streak);  withdraw 
needle;  replace  cotton  plug;  hold  needle  in  flame  until  glowing  red. — To 
prevent  the  sputtering  of  the  material  on  the  end  of  the  needle,  hold  near 
flame  until  dry  and  then  heat  to  redness.  Singe  free  exposed  end  of  the 
tube  cotton  plug  in  flame  to  kill  and  remove  microbes  and  spores  on  the 
outer  part  of  the  cotton.  The  inoculated  tubes  are  then  numbered  and 
incubated.  In  due  time  the  cultural  characteristics  are  noted  and  the 
observations  entered  in  a  suitable  note-book. 


PIG.  41. — Turck  ruling  of  the  Thoma-Zeiss  hemacytometer.  This  is  especially 
useful  if  it  is  desired  to  combine  the  bacterial  count  with  the  spore  and  yeast  count. 
The  smaller  areas  (1-400  sq.  mm.)  may  be  used  for  making  the  bacterial  counts,  while 
the  larger  areas  (1-25  sq.  mm.)  may  be  used  for  making  the  spore  and  yeast  counts. — 
(Carl  Zeiss.) 

Subcultures  may  also  be  made  in  Petri  dishes,  on  potatoes,  in  tubes 
containing  bouillon  broth,  blood  serum,  milk  and  other  media  with  or 
without  indicators. 

C.  Studying  Anaerobic  Microbes. — Some  microbes  have  anaerobic  tend- 
encies (facultative  aerobes)  and  some  are  absolutely  anaerobic  (obligative 
anaerobes).  The  deep  stab  culture  will  show  anaerobic  tendencies.  If 
such  tendency  exists,  development  will  be  more  active  near  the  bottom  of 
tube  (in  the  line  of  the  stab).  The  culturing  of  obligadve  anaerobes  re- 
quires special  apparatus  though  the  methods  are  not  in  any  way  difficult. 
The  following  methods  are  used: 

a.  Deep  stab  culture.     This  has  already  been  sufficiently  explained. 
It  merely  indicates  possible  anaerobic  tendencies. 

b.  High-culture  methods.     Fill  the  tube  of  a  deep  stab  culture,  shallow 
stab  or  streak,  with  liquid  agar  or  gelatin  and  incubate  in  the  usual  way. 
The  medium  to  be  poured  must  not  be  warmer  than  is  absolutely  necessary 


Il6  PHARMACEUTICAL  BACTERIOLOGY 

to  render  it  liquid.     This  brings  out  possible  anaerobic  tendencies  to  a 
more  marked  degree  than  does  the  simple  deep  stab  culture. 

c.  Make  an  Esmarch  roll  tube  culture  as  follows:  Roll  a  dilution 
gelatin  or  agar  tube  culture  (i  :  10,  i  :  100,  i  :  1000,  etc.)  so  that  all  of  the 
medium  (5  cc.  to  10  cc.)  is  spread  over  the  inner  surface  of  the  tube  to 
within  a  short  distance  of  the  cotton  plug.  Keep  on  rolling  slowly  until 
the  medium  has  set.  Roll  on  ice,  under  the  tap  water,  in  ice  water,  holding 
the  tube  at  the  proper  slant.  When  the  medium  has  set,  fill  in  the  entire 
tube  with  liquefied  gelatin  or  agar;  cool,  and  incubate.  Like  the  other 
methods  described,  this  will  show  possible  anaerobic  tendencies. 


FIG.  42. — Hanging-drop  culture,  sectional  profile  view.     These  slides  can  be  procured 
from  dealers  in  microscopical  supplies. 

d.  Various  methods  are  used  to  either  remove  the  air  (vacuum),  dis- 
place the  air,  or  remove  the  oxygen  from  the  air.  In  the  so-called  Buchner 
method,  potassium  hydroxide  and  pyrogallic  acid  are  used  to  take  up  the 
oxygen  of  the  air.  The  air  in  a  suitable  container  may  be  replaced  by 
hydrogen  by  means  of  a  Kipp  generator.  As  it  is  not  likely  that  the  phar- 
macist will  have  any  occasion  to  employ  these  methods  we  shall  pass  them 
by  with  this  mere  mention.  The  full  description  of  the  methods  will  be 
found  in  any  of  the  larger  works  on  medical  bacteriology  or  in  the  larger 
text-books  on  bacteriological  technic. 

D.  Microscopical  Examination  of  Microbes. — The  compound  micro- 
scope is  used  in  examining  hanging-drop  cultures,  water  mounts  and  cover- 
glass  preparations.  To  make  a  hanging-drop  culture,  hollow  ground  slides 
(concave  center)  are  required.  Touch  a  small  drop  of  the  culture  to  be 
examined  on  the  center  of  a  clean  and  heat-sterilized  cover-glass,  by  means 
of  a  heat-sterilized  platinum  wire  loop.  Smear  a  little  plain  petrolatum 
around  the  rim  of  the  concavity  of  the  slide  and  invert  the  cover-glass  prep- 
aration upon  the  slide,  pressing  it  gently  in  place  on  the  petrolatum. 
Examine  for  a  period  of  several  hours  or  longer  as  may  be  desired.  Cell 
division,  spore  formation,  etc.,  can  be  studied  very  conveniently.  Ob- 
servations on  the  effects  of  temperature  and  rate  of  septation  may  be 
made.  The  hanging-block  preparation  is  made  by  touching  the  surface  of 
a  cube  of  nutrient  agar  with  the  bacteria  and  then  applying  this  bacterial 
side  against  the  cover-glass  and  mounting  like  the  hanging  drop.  The 
bacteria  will  be  found  close  to  the  cover-glass. 

Bacteria  can  be  examined  mounted  in  water  on  a  slide  covered  with 
cover-glass,  in  order  to  make  observations  regarding  motility.  Of  course 
it  is  not  desirable  to  examine  pathogenic  microbes  in  this  manner  because 


BACTERIOLOGICAL   TECHNIC 


117 


of  the  possibility  of  infection.  In  any  case,  great  care  should  be  observed 
in  making  the  mounts.  The  slides,  covers  and  needle  used  must  be  steril- 
ized, every  antiseptic  precaution  must  be  observed;  and  avoid  placing  an 
excess  of  the  material  on  the  slide.  As  soon  as  the  observation  is  com- 
pleted (few  minutes  to  half  an  hour)  the  mount  (slide  cover  and  all)  should 
be  placed  in  a  5  per  cent,  solution  of  carbolic  acid  preparatory  to  cleaning. 


10  ^  T  —  ,7T, 


FIG.  43. — Jeffer's  circular  counting  plate  for  Petri  dish  cultures.     The  entire  area  (100 
sq.  cm.)  is  marked  off  into  ten  equal  sectors  of  ten  sq.  cm.  each. 

Cover-glass  preparations,  temporary  and  permanent,  are  made  as 
follows : 

a.  Clean  a  cover-glass  thoroughly,  dry  it  well  and  heat  it.  The  heating 
will  cause  the  smear  to  spread  better  and  to  adhere  better.  The  slides  to 
be  used  must  also  be  clean  and  dry. 


Il8  PHARMACEUTICAL  BACTERIOLOGY 

b.  By  means  of  the  platinum  needle,  spread  a  bit  of  the  bacterial 
growth  or  culture  over  the  greater  portion  of  the  surface  of  the  cover-glass. 
Add  a  droplet  of  water,  if  desired,  to  separate  the  bacteria  more.     Spread 
evenly.    Do  not  use  too  much  material,  as  it  will  make  an  unsightly 
mount. 

c.  Air-dry  the  smear  preparation.    This  requires  but  little  time,  per- 
haps a  minute  or  two. 

d.  Pass  the  cover-glass  preparation  through  the  flame  of  a  Bunsen 
burner  four  times.     This  must  not  be  done  too  slowly  as  that  will  char  or 
burn  the  microbes,  nor  yet  too  quickly,  as  that  would  not  coagulate  the 
albuminous  matter  and  thus  fail  to  fix  the  microbes  upon  the  cover-glass. 
A  little  experience  will  soon  teach  the  proper  speed.     Four  seconds,  or  a 
little  less,  is  the  average  time  in  which  to  make  the  four  passages  through 
the  flame. 

e.  Place  a  drop  or  two  of  the  stain  on  the  fixed  smear  and  allow  it  to 
act  long  enough  to  stain  sufficiently,  holding  the  cover-glass  over  a  flame  to 
warm  the  preparation.     Do  not  heat  it  more  than  60°  to  70°  C.     On  an 
average  the  stain  will  be  sufficiently  deep  in  five  minutes.     Fuchsin  re- 
quires longer  time  than  does  methyl-blue  or  gentian- violet. 

f.  Wash  off  the  excess  of  the  stain  under' a  small  hydrant  stieam  or  by 
means  of  a  wash  bottle,  or  by  moving  it  about  in  a  dish  of  water. 

g.  After  washing,  the  preparation  may  be  examined  as  a  temporary 
water  mount.     If  it  is  satisfactory  it  may  be  made  a  permanent  mount  by 
turning  the  cover-glass  up  again  and  allowing  the  water  to  evaporate  and 
then  mounting  in  Canada  balsam  with  xylene,  oil  of  cloves  or  some  other 
diluent  for  Canada  balsam.     Oil  of  cloves  acts  on  the  stain  for  which 
reason  xylene,  benzene  or  some  other  balsam  diluent  of  the  coal-tar  series 
is  preferable.     Special  staining  methods  have  already  been  explained. 
The  above  is  a  general  method  which  will  serve  most  purposes.     It  should 
be  kept  in  mind  that  the  staining  process  shrinks  the  microbes  somewhat. 
The  ordinary  staining  methods  do  not  bring  out  the  cilia.     The  fact  that 
the  microbe  is  motile  is  evidence  that  cilia  are  present,  though  it  cannot 
be  known  whether  they  are  unipolar,  bipolar  or  general,  single  or  multiple. 


CHAPTER  VII 

SYMBIOLOGY— THE  BIOLOGICAL  RELATIONSHIPS  OF 
ORGANISMS 

i.  GENERAL  INTRODUCTION 

Symbiology  is  the  science  which  treats  of  the  biological  relationship  of 
living  organisms,  animal  and  vegetable.  A  general  discussion  of  the 
subject  is  essential  to  a  clearer  comprehension  of  the  science  of 
bacteriology  or  microbiology.  It  will  be  found  that  in  the  treatment  of 
bacteria  in  disease  we  are  dealing  solely  with  symbiotic  relationships. 
The  term  symbiosis  has  been  variously  interpreted  and  misapplied. 
The  most  common  mistake  is  to  define  the  word  as  a  mutually  advan- 
tageous biological  or  physiological  association  of  two  or  more  organisms. 
Some  confuse  symbiosis  with  commensalism.  By  commensalism  is 
meant  the  living  together  of  two  or  more  parasitic  organisms  upon  a 
common  host,  and  is  therefore  *a  form  of  compound  symbiosis.  In 
this  case  the  common  (commensal)  parasites  apparently  bear  no  harmful 
relationship  to  each  other,  and  may  and  often  do  bear  a  mutualistic 
relationship  toward  each  other. 

Symbiology  in  the  broader  sense,  therefore,  means  the  science  which 
treats  of  parasitology,  of  symbioses  in  the  narrower  sense,  of  commensal- 
ism as  above  defined,  of  paracytosis,  of  patrocytosis,  of  leucocytosis,  and 
of  all  other  forms  of  intimate  physiological  and  pathological  associations 
of  living  things.  The  subject,  in  the  broader  and  more  comprehensive 
sense,  has  not  received  the  consideration  which  it  deserves,  although  some 
of  the  special  phases  have  been  very  fully  treated,  as  for  example  general 
parasitology  and  leucocytosis.  Nothing  more  can  be  done  than  to  outline 
the  subject  and  to  suggest  that  any  special  discussion  or  fuller  treatment 
be  looked  up  in  some  of  the  excellent  recent  monographs  (example — 
Herms,  William  B.  Medical  and  Veterinary  Entomology.  The  MacMil- 
lan  Company,  1915). 

A  statement  of  cytology  and  of  embryology  is  essential  to  the  better 
understanding  of  symbiology  and  the  related  sciences  adenology  and 
bacteriology.  The  following  is  a  brief  summary  of  the  subject. 

Van  Leeuwenhoek,  Hooke  and  Malphigi  (about  1660)  are  usually 
credited  with  having  made  the  first  observations  regarding  a  cellular 
structure  in  plants.  The  first  published  illustrations  of  plant  cells  per- 

119 


120  PHARMACEUTICAL  BACTERIOLOGY 

tained  to  cork  tissue.     Nothing  further  of  moment  was  done  until  about 
1833  when  Robert  Brown  again  took  up  the  study  of  the  cellular  elements 
of  plants.    Thus  it  will  be  seen  that  a  period  of  nearly  two  hundred  years 
had  elapsed  since  the  first  observations  on  plant  cells  during  which  no 
notable  progress  was  made  in  the  study  of  cytology.     This  long  period  of 
inactivity  was  no  doubt  occasioned  by  the  inadequacy  of  the  simple 
microscope  and  the  fact  that  the  compound  microscope  was  not  perfected 
sufficiently  to  be  of  any  great  advantage  over  the  simple  microscope,  until 
about  1825.     By  1838  and  1839  Schleiden  and  Schwann  were  ready  to 
formulate  a  cell  theory  of  living  things,  based  largely  upon  the  study  of 
plant  tissues  and  organs.     At  this  time  it  was  generally  believed  that  the 
cell- wall  represented  the  basic  or  essential  part  of  the  cell.     In  1846  von 
Mohl  called  attention  to  a  slimy  or  mucilaginous  substance  enclosed  by 
the  cell-wall  which  he  called  protoplasm,  believing  it  to  be  the  primal 
living  substance.     Dujardin,  Cohn  and  others,  now  began  to  give  some 
attention  to  the  animal  structure,  declaring  that  this  also  consisted  of 
cellular  elements  in  which  were  often  noticeable  certain  slimy  or  gelatinous 
substances  to  which  Dujardin  gave  the  name  sarcode.     Kolliker  noted  the 
fact  that  the  animal  cell  was  frequently  devoid  of  a  cell- wall.     Max 
Schultze  declared  that  the  protoplasm  of  the  plant  cell  and  the  sarcode  of 
the  animal  cell  were  in  all  essentials  alike.    Since  1870  a  multitude  of 
keen  observers  and  able  investigators  have  given  their  attention  to  the 
plant  cell  and  the  animal  cell  and  some  of  the  theories  based  upon  the 
discoveries  made  have  become  classics  in  scientific  annals.     To  review 
these  is  impracticable  and  unnecessary  for  the  present  purpose.     Atten- 
tion may  however  be  called  to  the  more  important  investigations  and 
theories. 

It  may  be  recalled  that  Harvey  in  the  i6th  century  declared  that  all 
life  came  from  an  egg  (Omne  vivum  ex  ovo)  which,  after  the  formulation  of 
the  cell  theory,  was  changed  to  the  declaration  that  every  cell  came  from 
a  preexisting  cell  (Omnis  cellula  ex  cellula),  and,  no  life  excepting  from  a 
previously  existing  living  organism  (Omne  vivum  e  vivo).  The  cell- wall 
lost  interest  and  the  entire  attention  was  now  centered  upon  the  cell- 
contents.  For  a  time  an  attempt  was  made  to  differentiate  between 
living  and  non-living  or  dead  cell  contents,  but  soon  the  conclusion  was 
reached  that  all  cell  parts  and  cell  constituents  were  the  product  of  plasmic 
activity  at  some  time  since  their  coming  into  existence.  Nor  did  it  take 
long  to  reach  the  conclusion  that  even  the  cell  was  not  the  ultimate  unit 
of  living  structure,  that  it  was  rather  a  living  complex  of  which  we  know 
very  little  and  of  which  we  cannot  know  very  much  until  the  chief  me- 
chanical aid  to  biologic  investigation,  namely  the  compound  microscope,  is 
more  highly  perfected  and  until  the  science  of  physical  and  biologic  chemis- 


SYMBIOLOGY — THE   BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS      121 

try  is  more  advanced.  Certain  text-books  still  persist  in  naming  and 
tabulating  the  physical  properties  of  the  cytoplasm;  for  example  stating 
that  it  is  viscid,  stringy,  slimy,  tenaceous,  semi-liquid,  semi-solid,  color- 
less, etc.,  etc.  It  may  be  recalled  that  so  eminent  an  investigator~as~ 
Biitschli  described  plasm  a  "  Wabenartig;"  others  that  it  was  fibrillated, 
or  granular^etc. 

It  may  be  recalled  that  Caspar  Wolff  advanced  the  theory  of  epigenesis 
or  the  development  of  individual  characteristics  through  environment, 
after  the  union  of  the  gametes  or  reproductive  cells.  Wolff  may  therefore 
be  considered  as  the  biological  champion  of  the  believers  in  the  formation  of 
character  through  environmental  influence.  In  1892  Weismann  formu- 
lated his  epochmaking  theory  regarding  the  continuity  of  the  germ  plasm, 
of  the  pre-f  ormation  and  the  pre-de termination  of  the  physical,  mental  and 
moral  traits  of  the  individual  which  were  supposed  to  be  held  or  bound 
within  the  reproductive  cells.  An  attempt  was  made  to  draw  a  sharp  line 
between  the  germ  cells  or  reproductive  cells  and  the  other  body  cells  or 
the  so-called  somatic  cells.  It  is  notable  that  the  major  theoretical  deduc- 
tions of  Weismann  have  in  the  main  been  proven  correct  in  the  light  of 
subsequent  cytologic  and  embryologic  investigations. 

Various  theories  were  advanced  with  a  view  to  explaining  the  mechan- 
ism of  the  transmission,  from  cell  to  cell  and  from  individual  to  individual 
of  the  inherent  or  hereditary  properties  and  characteristics  of  the  cell  and 
of  the  individual.  Darwin  very  ingeniously  assumed  the  existence  of 
biologic  molecules  which  he  called  gemmules.  It  was  supposed  that  each 
and  every  cell  possessed  the  biological  properties  of  every  other  cell  of  the 
body,  represented  by  the  gemmules.  Thus  it  was  assumed  that  a  kinetic 
secreting  cell  of  a  gland  for  example,  was  also  a  potential  nerve  cell;  or, 
that  any  somatic  cell  might  also  be  a  potential  germ  cell.  De  Vries  fol- 
lowed out  a  similar  line  of  reasoning  in  his  theory  of  pangenesis  in  which  he 
suggested  that  the  physical  carriers  of  the  hereditary  qualities  of  the  cell 
were  the  theoretically  assumed  pangenes  which  are  to  be  compared  to  the 
gemmules  of  Darwin.  Naegeli  advanced  the  micellar  theory  in  which  it  was 
assumed  that  certain  biologic  molecules  and  aggregates  of  such  molecules 
(the  micellae  and  the  pleons)  directed  or  controlled  the  growth  of  the  cell 
and  all  other  cell  activities.  Weismann  suggested  that  the  hereditary 
properties  of  the  germ  plasm  resided  in  the  idants  (composed  of  ids) 
which  were  also  theoretically  assumed  to  be  biologic  molecular  bodies, 
comparable  to  the  gemmules  of  Darwin  and  the  pangenes  of  de  Vries. 
The  structure  of  the  cell  and  its  many  constituents  received  attention, 
more  especially  the  nucleus  which  was  and  still  is  believed  to  be  the  essen- 
tial part  of  both  the  germ  cells  and  the  somatic  cells,  in  the  animal  as  well 
as  in  the  vegetable  organism.  The  chromatin  bodies  of  the  nucleus 


122  PHARMACEUTICAL  BACTERIOLOGY 

(chromosomes)  were  considered  to  be  the  carriers  of  and  the  transmitters  of 
the  hereditary  characters,  of  the  cell  and  we  have  come  to  believe  that  there 
can  be  no  new  cell  except  from  a  mother  nucleus,  at  least  as  far  as  pertains 
to  cells  which  have  nuclei.  While  it  is  generally  believed  that  the  germatic 
and  the  somatic  cells  have  distinctive  inherent  properties  which  are  not 
capable  of  being  transmitted  from  the  one  kind  of  cell  to  the  other,  there  are 
those  who  maintain  that  the  nucleus,  if  not  the  cell  plasm,  has  a  dual 
nature,  that  it  possesses  both  somatic  and  germatic  properties,  and  that 
the  somatic  cells  and  the  germatic  cells  are  therefore  potentially  inter- 
changeable. In  fact  some  investigators  have  suggested  the  possibility  of 
the  functional  interchangeability  of  somatic  and  germatic  cells  and  also 
of  the  male  and  female  germ  cells,  a  contention  which  has  been  proven  to 
be  correct  at  least  as  far  as  it  applies  to  the  lower  forms  of  plant  and  animal 
life.  Of  great  interest  have  been  the  recent  experiments  in  ovarian  and 
testicular  transplantation  made  by  Castle,  Stanley  and  others,  the  results 
of  which  tend  to  prove  the  correctness  of  the  Weissmannian  theory  of  the 
transmission  of  hereditary  qualities.  For  example,  the  Guinea  pig  off- 
spring derived  from  a  transplanted  ovary  possessed  the  qualities  of  the 
mother  from  which  the  ovary  was  taken  and  not  those  of  the  pig  into  which 
the  ovary  had  been  transplanted.1  We  must  not  forget  the  epochmaking 
experiments  of  the  Austrian  monk  Mendel  (1822—1884)  and  the  Mendelian 
law  of  the  transmission  of  hereditary  qualities.  Mendel  demonstrated 
that  the  gametic  fusion  of  different  ancestral  characteristics  did  not  give 
rise  to  a  blend  of  such  characteristics,  but  rather  that  there  was  a  continua- 
tion of  the  hereditary  qualities  of  both  gametes  in  dominant  and  recessive 
proportions.  It  is  surprising  that  this  fundamental  principle  or  rule  in 
gametic  or  sexual  reproduction  was  not  noted  earlier.  It  must  have  been 
apparent  to  all  observers  that  the  combining  of  male  and  female  hereditary 
sex  characteristics,  as  must  be  the  case  in  every  gametic  fusion,  did  not  as 
a  rule  result  in  a  hermaphrodite  or  sexually  neuter  being.  The  child  does 
not  inherit  a  blend  of  paternal  and  maternal  characters.  A  son  may  in- 
herit the  physical  characters  from  the  maternal  side  of  the  house  and  the 
mental  and  moral  characters  from  the  paternal  side.  The  offspring  of  an 
ill  tempered  and  an  indifferent  parent  does  not  grow  into  an  even  or 
happily  tempered  person.  Both  characteristics  may  be  present,  one 
dominant  and  the  other  recessive  but  not  as  a  blend.  In  this  connection 
might  be  mentioned  the  theory  of  male  dominance  as  promulgated  by 
Galton  and  others.  That  is,  the  offspring  manifests  to  a  dominant 
degree  the  paternal  hereditary  qualities.  There  are  however  numerous 
exceptions  to  this  rule. 

1  Experimental  tissue  transplantations,  tumor  transplantations  and  grafting,  show 
similar  results. 


SYMBIOLOGY — THE   BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS      123 

Prior  to  the  discovery  of  the  cellular  structure  of  plants  and  animals, 
there  was  some  discussion  and  theorization  as  to  an  ultimate  unit. of  living 
structure;  that  is,  what  might  be  the  smallest  part  of  a  higher  plant,  for 
example,  which  would  continue  to  exist  and  mature.  Many  held  that  the 
phyton  or  phytomer  (node,  i.e.,  node  and  internode)  was  the  smallest  plant 
part  which  would  continue  to  exist  vegetatively  and  develop  into  a  new 
mature  individual.  It  is  however  known  that  even  smaller  cell  aggre- 
gates of  a  plant  can  be  induced  to  form  a  new  individual,  as  leaf  and  part 
of  leaf  (Begonias),  bits  of  rhizome,  of  tubers  and  of  stems.  In  the  case  of 
the  lower  plants  and  animals  the  reduction  can  even  be  carried  to  the 
individual  cell.  The  Saccharomycetes  are  probably  multicellular  plants 
in  the  process  of  formation  and  in  this  group  any  single  cell  is  capable  of 
forming  new  cell  aggregates  by  the  budding  process.  In  the  class  Spon- 
gilla  a  group  of  a  few  cells  will  form  a  new  sponge  mass.  In  the  lichens, 
lower  algae,  fungi,  and  liverworts,  small  bits  representing  a  few  cells  will 
mature  into  new  individuals. 

With   the  advent  of  the  cell  theory  the  immediate  conclusion  was 

reached  that  the  cell  represented  the  ultimate  unit  of  living  structure 

but  as  already  stated  we  now  know  that  the  cell  itself  is  an  aggregate 

of  different  kinds  of  more  or  less  highly  differentiated  living  units.     In  the 

lower  forms,  as  amebae,  paramecia,  bell  animalculae,  etc.,  the  cell  may  be 

divided  mechanically  and  the  several  fragments  will  each  develop  into  a 

new  mature  cell.     Certain  plastids,  nuclear  chromosomes  and  plasmic 

granules  (plasomes,  chondriosomes,  etc.)  will  live  for  a  time  outside  of  the 

cell,  in  water,  in  isotonic  solutions  and  in  the  presence  of  certain  active 

plant  constituents  (caffein,  asparagin).     Tissues  and  organs  have  been 

transplanted  from  one  animal  to  another  (skin  grafting,  bone  grafting, 

ovarian  grafting,  cancer  grafting,  etc.)  and  certain  tissues  have  been 

induced  to  grow  in  artificial  culture  media   (epithelial  cells,  muscular 

tissue,  etc.,  in  blood  plasm)  but  no  one  has  as  yet  succeeded  in  developing 

a  higher  plant  or  animal  from  a  single  detached  somatic  cell,  nor  has  any 

one  succeeded  in  perpetuating,  in  artificial  media,  any  of  the  living  cell 

inclusions.     There  appears  to  be  no  difficulty  in  keeping  groups  of  living 

tissue  cells,  which  are  kept  in  an  undisturbed  trophic  relationship,  alive 

for  considerable  periods  of  time,  but  to  induce  such  cell  aggregates  to  grow 

and  to  multiply  by  septation  is  apparently  more  difficult.     Fruits,  seeds, 

eggs,  ova  and  some  larval  forms,  will  remain  viable  for  long  periods  under 

natural  and  also  under  certain  artificially  maintained  conditions.     The 

cells  and  certain  plasmic  cell  inclusions  of  the  apple,  the  grape,  the  melon, 

the  pumpkin,  the  tomato,  etc.,  will  remain  alive  until  decomposition  sets 

in  or  until  the  loss  of  moisture  becomes  excessive.     Sections  of  fleshy 

roots,  tubers,  fruits  and  rhizomes  mounted  in  water  or  in  isotonic  solutions 


124  PHARMACEUTICAL  BACTERIOLOGY 

will  show  living  cell  elements  which  may  be  studied  for  many  hours  and 
even  days,  and  some  of  the  plastids,  chromophores  and  nuclei,  will  not 
only  remain  alive  for  weeks  when  suspended  in  the  hanging  drop  but 
will  also  increase  in  size  and  in  rarer  instances  will  multiply  by  septation. 

We  have  from  the  first  recognized  and  admitted  the  transmissibility 
of  the  parental  characteristics  via  the  male  and  female  germ  cells,  and  such 
hereditary  transmission  is  undeniable.  In  order  to  explain  the  phyloge- 
netic  and  ontogenetic  relationship  of  the  somatic  cells  and  the  germatic 
cells,  we  must  survey  our  present  conception  of  the  origin  of  the  gametes 
or  the  sexual  reproductive  cells. 

]We  may  assume  that  the  original  living  structures  or  organisms  con- 
sisted of  individualized  bits  of  more  or  less  complexly  differentiated 
plasmic  substances  and  we  may  also  assume  that  these  plasmic  units  were 
biologically  somatic  or  trophic  in  character  rather  than  germatic  or  re- 
productive. We  can  readily  comprehend  why  in  the  very  nature  of  things 
these  living  bits  of  plasm  were  self  limited  as  to  size  and  also  as  to  duration 
of  existence,  in  all  probability  largely  due  to  the  limitations  of  available 
food  materials  in  the  immediate  vicinity  of  the  originally  motionless 
plasmic  units. 

The  essential  of  reproduction  is  the  detaching  of  a  single  cell  or  a  group 
of  cells  from  the  parent  cell  or  parent  body,  capable  of  continued  existence, 
and  growth.  Three  types  of  reproduction  are  generally  recognized,  vege- 
tative reproduction,  spore  reproduction  and  sexual  or  gametic  reproduc- 
tion. In  the  lower  plants  and  animals  all  three  methods  may  be  observed. 
In  the  alga  Ulothrix,  for  example,  a  portion  of  the  vegetative  filament 
may  become  detached  and  such  detached  cell  or  group  of  cells  will  continue 
growth  and  septation  and  finally  develop  into  a  new  mature  filament.  As 
the  conditions  for  the  purely  vegetative  method  of  reproduction  became 
more  and  more  unfavorable,  the  contents  of  certain  specialized  cells  of  the 
filament  were  formed  into  spores  which  possess  the  unusual  quality  of 
being  able  to  tide  over  an  unfavorable  period.  In  time  even  the  spore 
was  no  longer  sufficient  to  enable  the  organism  to  survive  the  changing 
environmental  conditions  and  the  gametic  method  was  developed.  There 
certainly  can  be  no  doubt  as  to  the  priority  of  the  vegetative  method  of 
reproduction  as  represented  by  the  simpler  forms  of  cell  septation  and  by 
budding  and  the  renewed  growth  of  detached  cell  groups.  In  sexual  re- 
production we  have  the  union  or  fusion  of  the  chromosomes  and  perhaps 
other  essential  elements  of  two  different  cells  (male  and  female  gametes) 
of  the  same  species.  Since  the  somatic  or  vegetative  cell  and  the  vegeta- 
tive methods  of  reproduction  preceded  the  gametes  and  the  sexual  method 
of  reproduction,  we  must  assume  that  gametes  are  somatic  in  origin,  both 
phylogenetically  and  ontogenetically. 


SYMBIOLOGY — THE  BIOLOGICAL  RELATIONSHIPS    OF    ORGANISMS     125 

It  is  generally  admitted  that  gametic  or  sexual  reproduction  secures 
or  attains  a  union  or  fusion  of  extreme  variations  of  living  cells  which 
resulted  from  the  variations  in  environmental  influence,  which  union  or 
fusion  produced  an  optimum  adaptability  to  the  causative  variations~irr 
the  environment.  Let  us  suppose  that  two  groups  (I  and  II)  of  unicellular 
organisms,  perhaps  of  the  amebic  type,  should  by  chance  be  placed  in 
different  environments,  group  I  in  a  medium  with  an  ample  food  supply, 
and  group  II  in  a  medium  with  insufficient  food.  The  individual  cells  of 
group  I  would  grow  comparatively  larger  and  become  more  sluggish  and 
inactive.  The  individuals  of  group  II,  because  of  insufficient  food  would 
become  smaller  and  develop  increased  motility  for  the  purpose  of  securing 
more  of  the  scant  food.  As  a  result  of  this  environmental  difference 
there  would  in  time  result  two  sets  of  living  cells  derived  from  one  and  the 
same  species  which  would  differ  morphologically  and  physiologically. 
Should  these  two  groups  be  compelled  to  continue  in  these  environments, 
death  or  extinction  would  probably  follow,  on  the  one  hand  because  of 
hypernutrition  and  resulting  inertia,  and  on  the  other  hand  because  of  in- 
sufficient food.  We  can  imagine  the  condition  of  one  or  more  of  the 
smaller  and  starving  but  more  active  cells  finding  one  or  more  of  the  hyper- 
nourished  and  inactive  larger  cells  and  seizing  upon  these  primarily  for 
the  purpose  of  securing  a  food  supply.  This  attack  on  the  part  of  the 
smaller  cell  would  probably  result  in  more  or  less  reaction  in  the  larger 
cell.  Perhaps  the  withdrawal  of  some  of  the  proteid  excess  restored  or 
awakened  or  aroused  some  of  the  lost  energy.  In  brief,  the  biological 
association  of  these  different  cells  proved  mutually  beneficial.  It  is 
reasonable  to  suppose  that  such  primal  gametoid  association  should  be 
temporary,  rather  than  a  permanent  fusion,  as  in  the  gametic  cell  fusion 
in  higher  plants  and  animals.  Such  temporary  gametoid  cell  associations 
occur  in  the  group  Paramecium,  and  represents  the  lowest  or  least  special- 
ized form  of  sexual  reproduction.  In  the  illustration  cited,  the  larger 
cell  might  be  considered  the  female  gamete  and  the  smaller  one  the  male 
gamete.  There  are  gametes  which  appear  to  be  identical  as  to  size,  form 
and  color,  but  it  is  irrational  to  suppose  that  such  germatic  cells  are  identi- 
cal physiologically  and  chemically.  We  cannot  imagine  what  might  be 
the  gain  in  the  gametic  union,  either  temporary  or  permanent,  of  two  iden- 
tical cells.  In  other  words  it  is  extremely  doubtful  if  there  are  genuinely 
isogamous  plants  or  animals. 

We  can  illustrate  the  advantages  resulting  from  the  union  of  the 
properties  of  two  gametes  by  the  classical  chart  by  Wilson.  We  may  rep- 
resent the  properties  of  the  originally  somatic  cells  by  the  first  four  letters 
of  the  alphabet.  In  one  group  of  cells  the  properties  developed  to  the 
maximum  (A,  B,  C,  D),  in  the  second  group  the  same  properties  were 


126 


PHARMACEUTICAL  BACTERIOLOGY 


reduced  to  a  minimum  (a,  b,  c,  d).  In  the  game  tic  union  these  properties 
would  be  combined  in  the  zygote,  at  least  we  can  assume  that  such  might 
be  the  case.  In  the  cell  septations  which  would  result  from  this  zygote 
the  properties  (inherited)  of  the  two  gametes  might  appear  in'the  daughter 
cells  in  sixteen  possible  combinations  and  it  is  readily  comprehensible 
how  and  why  some  of  the  combinations  would  be  more  suitably  adapted 
to  the  environment  than  others  and  these  would  secure  the  survivalfof  ih* 
race  or  species. 


FIG.  44. — Illustrating  the  redistribution  of  hereditary  properties  after  the  fusion 
of  the  female  gamete  (I)  and  the  male  gamete  (II),  forming  the  zygote  (Z),  which 
upon  starting  a  new  septating  cycle  gives  rise  to  cells  in  which  the  original  properties 
of  the  two  gametes  (A,  B,  C,  D,  and  a,  b,  c,  d)  may  be  rearranged  in  sixteen  different 
combinations.  (Adapted  from  the  chart  by  Wilson.) 

The  investigations  of  Butschli,  Calkins,  Hertwig,  Jennings,  Maupas 
and  others,  led  to  the  conclusion  that  the  union  of  gametes  in  sexual  repro- 
duction had  the  effect  of  adapting  a  few  out  of  many  to  the  environment, 
through  restoration  or  augmentation  or  restimulation  of  weakened  ac- 
tivities. The  somatic  existence  of  single-celled  animals  and  of  the  many- 
celled  animals,  has  occasioned  much  discussion.  It  has  been  customary  to 
speak  of  single-celled  animals  such  as  the  paramecia,  the  amebas,  etc.,  as 
endowed  with  eternal  life,  barring  accidents,  and  that  the  complex  organ- 
isms as  man,  for  example,  is  doomed  to  suffer  an  unavoidable  death  of 
the  somatic  body.  No  such  difference  exists  if  we  draw  the  correct 
biological  parallellism.  If  we  compare  the  life  cycle  of  the  single-celled 
organism  in  a  given  medium,  with  the  life  cycle  of  the  cells  of  the  body  of  a 
higher  organism,  we  note  a  very  close  analogy.  If  we  inoculate  a  given 


SYMBIOLOGY — THE   BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS      127 

medium  with  a  given  species  of  paramecium  and  keep  the  food  supply 
constant  and  uniform,  we  find  that  sooner  or  later  the  organisms  gradually 
divide  or  septate  less  actively  until  finally  all  septa tion  ceases  and  the 
medium  becomes  freed  of  living  paramecia.  In  other  words,  the  organ- 
ism has  completed  its  cycle  of  somatic  existence,  which  may  have  lasted 
hundreds  and  thousands  of  generations.  By  means  of  certain  stimuli,  as 
strychnine,  alcohol,  etc.,  the  cycle  may  indeed  be  prolonged  by  many 
additional  generations  but  even  these  stimuli  will  not  make  it  possible  to 
maintain  the  cycle  indefinitely.  In  the  case  of  bacteria,  biological  cycles 
have  been  maintained  for  many  years,  apparently  without  appreciable 
diminution  of  the  septating  powers  of  the  cells,  but  bacteriologists  declare 
that  sooner  or  later  a  strain  of  bacteria  will  deteriorate  and  take  on  ir- 
reparable changes.  In  a  similar  manner  the  somatic  cells  of  a  many- 
celled  animal  complete  the  biological  cycle  and  somatic  death  follows. 
In  the  one  case  the  individual  cells  are  apparently  all  alike  and  live  apart, 
and  in  the  second  case  the  individual  cells  differ  morphologically  as  well  as 
physiologically  and  are  united  by  growth  and  have  become  specialized 
into  tissues  and  organs.  Just  as  the  continued  existence  of  the  single- 
celled  race  is  made  possible  through  the  gametic  relationships  of  certain 
cells  just  so  the  life  of  the  somatic  cells  of  higher  plants  and  animals  is 
made  continuous  through  the  reproductive  cells. 

Within  recent  years  many  researches  have  been  made  in  cytology  and 
embryology.  Of  these  the  most  interesting  were  the  discovery  of  the  sex 
chromosomes  and  the  relationship  of  these  bodies  in  the  male  and 
female  gametes,  by  Boveri,  Henking,  McClung,  Wilson  and  others.  The 
chemism  of  the  egg  cell  received  the  attention  of  Baltzer,  Herbst,  Loeb  and 
many  others.  It  was  found  that  the  egg  cell  of  the  sea  urchin,  of  the  frog 
and  of  other  animals,  could  be  induced  to  segment  without  the  presence  of 
the  male  cell  (Herbst,  Loeb).  The  most  spectacular  discovery  was  that 
of  the  sex  determinant  in  the  chromosomes  of  the  male  or  sperm  cell.  It 
was  found  that  the  germ  cells  of  higher  plants  and  animals  differ  from  the 
somatic  cells  in  the  reduction  of  the  chromosome  bands  (24  in  the  somatic 
cells  as  against  1 2  in  the  germatic  cells,  as  a  rule) .  The  1 2  chromosomes 
of  the  female  germ  cell  fuse  making  6  larger  chromosomes.  In  the  male 
germ  cell  a  most  remarkable  irregularity  in  the  union  of  the  chromosomes 
was  observed,  upon  which  irregularity  the  determination  of  sex  depends. 
Some  of  the  sperm  cells  contain  1 2  chromosomes  and  an  extra  larger  chro- 
mosome, which  is  the  sex  determining  chromosome.  There  are  therefore 
two  kinds  of  male  reproductive  cells,  one  with  six  fused  chromosomes  and 
the  other  with  seven  chromosomes  (six  fused  and  one  single) .  The  latter 
are  the  male  producing  sperm  cells.  The  male  and  female  producing 
sperm  cells  are  supposed  to  be  present  in  about  equal  numbers.  Which 


128  PHARMACEUTICAL  BACTERIOLOGY 

kind  of  sperm  cell  will  fuse  with  the  egg  cell  is  manifestly  a  matter  of 
chance.  In  some  animals  the  male  sex  determinant  is  a  double  or  fused 
bu  I  larger  chromosome  and  again  the  male  sex  chromosome  may  be  double 
but  remain  unfused  or  uncombined.  It  is  of  interest  to  note  that  while 
the  male  cell  is  as  a  rule  much  smaller  than  the  female  and  suffers  practi- 
cally a  complete  loss  of  identity  at  the  time  of  fertilization  of  the  egg  cell, 
it  is  nevertheless  the  dominant  factor  in  the  transmission  of  hereditary 
qualities. 

We  may  sum  up  the  essentials  of  our  present  knowledge  of  cytology 
as  follows: 

1 .  The  somatic  cell  preceded  the  germ  cell,  and  all  living  cells  and  cell 
units  must  have  been  derived  from  a  plasm  of  somatic  or  trophic  character. 

2.  Gametes  (both  male  and  female)  are  merely  differentiated  somatic 
cells  of  the  same  kind  and  are  the  product  of  extreme  variations  in  the 
environment.     Germatic  cells  differ  from  somatic  cells  in  the  reduced 
chromosomes. 

3.  Germatic  chromosomes  as  well  as  somatic  chromosomes  are  living 
plasmic  complexes  derived  from  the  cell  plasm.     The  plasm  in  all  proba- 
bility consists  of  and  contains   the  ultimate  units  of  living  structure 
which  ultimate  units  are  the  creators  and  generators  of  the  living  formed 
cell  constituents  of  all  kinds. 

4.  Gametes  have  the  property  of  combining  or  fusing  certain  sub- 
stances which  are  believed  to  be  more  or  less  essential  to  the  continued 
septation  of  the  egg-cell.     Loeb  and  others  have  proven  experimentally  that 
the  male  germ  cell  is  not  absolutely  essential  to  embryonal  development. 

5.  The  mitotic  (karyokinetic)  changes  in  the  somatic  cells  manifest- 
some  of  the  characteristics  of  the  fusion  of  the  game  tic  chromosomes.     The 
biological  and  physiological  activities  of  the  cell  (microcosm;  are  very 
closely  analogous  to  the  activities  of  the  cells  forming  the  body  of  the 
individual  (macrocosm). 

6.  The  biological  association  (symbiosis)  of  cells  of  the  same  somatic 
origin  (resulting  from  septation)  in  the  multicellular  organism,  is  an  evolu- 
tional incident,  probably  occasioned  by  the  variations  in  the  environment. 
Associations  of  cells  of  this  kind  are  in  process  of  formation  at  the  present 
time  among  the  Saccharomycetes,  the  group  bacteria,  the  lower  algae  and 
among  the  lower  groups  of  the  animal  kingdom. 

7.  We  can  readily  comprehend  how  the  originally  homogeneous  cells 
resulting  from   septation,   composing   the  complex  organism,  gradually 
differentiated  into  tissues  and  organs  each  endowed  with  specialized 
activities  and  functions. 

8.  There  are  indications  however,  that  the  cells  of  certain  organisms 
are  of  heterogeneous  origin.     In  the  water  net  (Hydrodictyon)  for  exam- 


SYMBIOLOGY — THE  BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS      I2Q 

pie,  the  cells  are  unquestionably  of  homogeneous  origin;  whereas  in  the 
lichen  group  we  know  for  a  certainty  that  the  cells  are  of  heterogeneous 
origin  (alga  and  fungus).  It  is  highly  probable  that  some  of  the  living 
elements  of  the  cells  of  higher  plants  are  of  heterogeneous  origin,  like-" 
wise  those  of  such  animals  as  the  chlorophyll  bearing  Hydra  mridis  and  the 
chlorophyll  bearing  amebae. 

9.  Whether  or  not  the  cells  resulting  from  septa tion  remain  free  or 
uncombined  as  in  the  protozoa,  bacteria,  single-celled  algae,  etc.,  or  united 
as  in  many-celled  plants  and  animals,  is  incidental  in  the  order  of  evolu- 
tion.    In  both  cases  septation  is  cyclical,  that  is,  it  continues  until  the 
septating  power  is  exhausted  and  death  of  the  entire  somatic  cell  associa- 
tion follows.     In  both  cases  extinction  of  the  cyclical  and  individualistic 
and  autonomous  cell  groupings  is  prevented  by  spore  formation  and  by 
the  gametic  fusion  of  certain  specialized  cells. 

10.  All  evidence  points  to  the  biological  fact  that  as  the  association 
of  cells  of  the  same  kind  became  closer  and  closer  physically,  there  was 
developed  a  corresponding  increase  in  the  biological  and  physiological 
interdependence  of  the  cells,  with  a  gradual  reduction  or  lessening  of  the 
individualism  or  independence  of  the  cells.     In  the  lowest  many-celled 
plants  and  animals,  a  single  cell  still  retains  the  power  to  develop  into  a 
new  individual,  as  in  Saccharomyces,  diatoms,  desmids,  water  net,  lower 
filamentous  algae,  streptococci,  etc.     As  the  cell  grouping  became  more  and 
more  intimate  and  interdependent  biologically,  the  individual  cell  could 
no  longer  dissociate  itself  and  develop  into  a  new  group.     The  individual 
cell  can  only  septate  while  in  contact  or  biological  association  with  its 
fellows.     Fragments  representing  a  variable  number  of  cells  still  in  biologi- 
cal association,  of  certain  lower  plants  and  animals,  as  among  the  algae, 
the  fungi,  the  lichens,  hydras,  sponges,  liverworts,  etc.,  etc.,  could  still 
develop  into  new  organisms.     Finally  the  somatic  cells  of  the  organism 
lost  wholly  the  power  of  developing  into  a  new  individual,  no  matter  how 
large   the   dissociated  fragment  might  be,   as  is  the  case  in  all  higher 
animals  and  in  many  of  the  higher  plants. 

1 1 .  The  living  inclusions  of  the  cell  are  even  more  intimately  interde- 
pendent biologically  (somatically)  than  are  the  cells  composing  the  indi- 
vidual, and  it  is  therefore  not  surprising  to  find  it  very  difficult,  if  not 
absolutely  impossible,  to  induce  such  plasmic  cell  units  to  multiply  when 
dissociated  form    the   mother   cell.     Amyloplastids,   chloroplastids   and 
leucoplastids  have  been  seen  to  increase  numerically  outside  of  the  cell  in 
the  hanging  drop  but  so  far  no  one  has  succeeded  in  inducing  such  nu- 
merical increase  to  proceed  to  any  very  considerable  cell  mass  formation. 
The  indications  are  that  any  such  numerical  increase  is  more  apparent 
than  real.     That  is,  the  plastids  which  were  already  present  in  the  embryonic 


130  PHARMACEUTICAL  BACTERIOLOGY 

stage  at  the  time  the  hanging  drop  was  prepared  simply  grow  to  maturity, 
without  actual  numerical  increase.  There  are  however  indications  that 
there  is  also  actual  increase  by  septation  or  by  extension  from  the  cell 
plasm. 

That  the  fusion  of  gametes  is  intimately  bound  up  with  the  activities 
and  evolutional  development  of  the  somatic  cells,  is  indicated  by  the  fact 
that  only  those  gametes  which  are  derived  from  very  closely  related  organ- 
isms, will  combine  sexually. '  While  it  is  quite  evident  that  gametic  repro- 
duction is  the  product  of  environmental  influences  which  have  been  at 
work  for  ages,  the  why  and  wherefore  of  such  reproduction  is  not  clear. 
To  state  that  certain  proteid  stimulins  or  perhaps  enzymatic  bodies, 
such  as  fertilizin,  spermin,  etc.,  are  concerned  in  sex  fusion  and  in  sex 
differentiation,  does  not  materially  clear  the  situation,  unless  we  can  explain 
how  and  why  these  substances  produce  the  effects  ascribed  to  them. 
Sex  evolution  still  is  and  for  some  time  to  come  will  continue  much 
of  a  mystery. 

We  may  assume  that  it  is  an  established  fact  that  the  somatic  cell 
preceded  the  germatic  cell  and  that  formed  and  living  cell  inclusions,  such 
as  the  chromoplasteds,  chloroplastids,  amyloplastids,  leucoplastids,  chro- 
mosomes, directive  spheres,  chondriosomes,  mitochondria,  etc.,  have 
their  origin  (phylogenetically  as  well  as  ontogenetically)  in  the  cell  plasm, 
probably  derived  from  fusing  and  septating  plasmic  granula  and  from 
other  as  yet  unrecognized  ultimate  plasmic  elements.  We  are  therefore 
also  justified  in  assuming  that  the  essentials  of  cell  growth  and  of  reproduc- 
tion reside  in  the  plasm  of  the  somatic  cell  and  that  mitosis  which  for  several 
decades  has  been  looked  upon  as  the  dominant  and  all-important  cell 
activity,  is  of  secondary  significance. 

We  have  indicated  the  genetic  relationship  of  somatic  and  germatic 
cells,  of  single-celled  and  many-celled  organisms,  of  cells  and  of  cell  contents, 
of  cell  plasm  and  of  chromosomes.  It  is  believed  that  the  observations  of 
sphaerocytes  is  further  conclusive  evidence  that  the  cell  plasm  is  the  source 
of  all  living  inclusions  and  that  the  plasm  of  the  somatic  cell  of  higher  organ- 
isms may  under  certain  conditions,  produce  not  only  a  single  cell  but  also 
an  indefinite  number  of  secondary  cells.  This  is  evident  from  the  study 
of  plant  sphaerocytes. 

The  sphaerocytes  of  plants  are  living  structures  derived  from  the  cell 
plasm,  possessing  the  characteristics  of  the  cells  from  which  they  are 
derived.  They  occur  largely  extracellular,  that  is,  outside  of  the  mother 
cell  from  which  they  originated,  although  in  some  fruits,  as  the  grape  and 
the  tomato,  many  are  found  within  the  cell.  In  the  squash  they  appear 
to  be  very  largely  if  not  wholly  intra-cellular.  In  size  they  are  extremely 
variable,  ranging  from  the  limits  of  microscopic  vision  to  over  100  microns 


SYMBIOLOGY — THE   BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS      131 

in  diameter.  The  normal  form  of  the  sphaerocytes,  as  the  name  would 
indicate,  is  that  of  a  perfect  sphere,  although  some  are  more  or  less  irregular 
in  outline  and  some  show  marked  ameboid  movement.  Actively  motile 
sphaerocytes  are  comparatively  numerous  in  the  green  tomato  and  ateo 
in  the  green  grape.  The  smaller  and  probably  the  younger  sphaerocytes 
of  the  fruits  thus  far  examined  have  the  following  characteristics  in 
common: 

i.  They  are  spherical  in  form  excepting  the  motile  forms  referred  to. 


FIGS.  45~48b   x  450. 

FIG.  45. — Different  forms  of  sphaerocytes  from  the  mucilaginous  tissue  of  tomato 
seeds.  A,  a  fully  matured  sphaerocyte  cell  (Gliding  cell);  a,  nucleus  with  nucleolus; 
b,  reddish  brown  coloring  granules;  c,  intra-cellular  sphaerocytes;  d,  chlorophyll  gran- 
ules. B,  sphasrocyte  nearly  mature,  showing  several  endo-sphaerocytes.  C,  sphaero- 
cytes in  various  stages  of  development.  D,  amebo- sphaerocytes  of  the  ripe  tomato. 
E,  a  mature  nucleo-sphaerocyte;  a,  nucleus  with  nucleolus;  b,  reddish  brown  coloring 
particles;  c,  vacuoles;  d,  granular  cell-plasm. 

2.  The  plasmic  contents  are  rather  delicately  granular  but  there  is 
no  evidence  of  streaming  plasmic  motion.     The  plasmic  granula  often  show 
slight  active  and  also  Brownian  motion. 

3.  There  is  no  outer  limiting  membrane  or  cell  wall,  at  least  none  is 
demonstrable  by  the  usual  staining  methods. 

4.  Vacuoles  are  generally  present  and  are  very  variable  in  size.     In 
some  sphaerocytes  a  single  vacuole  may  occupy  the  greater  portion  of  the 
cell,  leaving  a  mere  meniscoid  outer  rim  of  granular  plasm.     More  gener- 


132 


PHARMACEUTICAL  BACTERIOLOGY 


ally  there  are  two  or  more  smaller  vacuoles,  sometimes  as  many  as  twenty 
or  thirty,  again  there  may  be  one  comparatively  large  vacuole  and  two  or 
more  smaller  vacuoles. 

5.  There  is  a  striking  similarity  between  the  younger  sphaerocytes  as 
above  described  and  the  resting  or  encysted  forms  of  amebae.     In  fact 
so  striking  is  this  resemblance  that  it  was  at  first  supposed  that  they 
might  be  stages  in  the  life  history  of  certain  amebae,  but  further  obser- 
vation proved  this  supposition  erroneous.     Sphaerocytes  disappear  with 
the  advent  of  decomposition  whereas  amebae  thrive  in  the  presence  of 
decaying  substances. 

6.  The  colorless  younger  sphaerocytes  are  generally  without  nuclei;  at 
least  none  could  be  detected  by  the  usual  staining  methods. 


FIG.  46. — Amebo-sphaerocytes  of  the  green  tomato.  A,  actively  motile  forms  with 
chlorophyll  granules.  B,  actively  motile  forms  without  chlorophyll  granules.  C 
encysted  amebo-sphaerocytes  with  chlorophyll  granules.  D,  encysted  forms  without 
chlorophyll  granules. 

The  sphaerocytes  are  very  abundant  in  the  mucilaginous  layer  enclosing 
the  seeds  of  the  tomato  (Schleimhiille),  where  they  occur  in  all  stages 
of  development,  ranging  in  size  from  the  very  limits  of  microscopic  iden- 
tification (about  one  micron  in  diameter)  to  mature  mucilaginous  tissue 
cells  (275  microns  in  diameter).  They  originate  in  the  cell  plasm  and  are 
soon  extruded  from  the  cell  plasm  whereupon  they  continue  an  independent 
existence  within  or  without  the  mother  cell.  The  extra-cellular  forms  no 
doubt  make  their  escape  from  the  cell  by  way  of  the  pores  of  the  cell- wall. 
Some  may  be  derived  from  intercellular  plasmic  threads. 

Occasionally  groups  or  clusters  of  sphaerocytic  pulp  cells  occur  in  cer- 
tain areas  of  the  tomato  pulp,  more  especially  near  the  epidermal  layers. 
It  would  appear  that  under  ordinary  or  usual  conditions  the  sphaerocytes 


SYMBIOLOGY — THE  BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS      133 

are  formed  sparingly,  or  at  least  they  do  not  develop  to  larger  size  in  con- 
siderable numbers.  Due  to  the  action  of  certain  stimuli  they  may  develop 
very  rapidly  in  large  numbers,  forming  a  new  or  an  additional  tissue  iden- 
tical with  or  very  closely  similar  to  the  tissue  from  which  they  had  their 
origin.  In  other  words  sphaerocytes  may  give  rise  to  a  neoplasmic  growth, 
of  which  the  mucilaginous  layer  enclosing  the  seed  of  the  tomato  is  a 
striking  example. 

The  following  different  kinds  of  sphaerocytes  may  be  recognized  in  the 
tomato.  These  are,  in  all  probability,  different  stages  in  the  development 
of  a  sphaerocyte. 

a.  Leuco-spharocytes. — These  are  the  youngest  and  earliest  stages  in 
the  development  of  sphaerocytes,  as  has  already  been  explained.     They 
usually  range  from  one  micron  to  about  six  microns  in  diameter. 

b.  Amebo-sphcerocytes. — These  resemble  the  leuco-sphaerocytes  in  that 
they  are  colorless  and  contain  vacuoles.     They  differ  in  that  they  are  al- 
ways irregular  in  form  and  show  a  very  slow  to  a  rather  marked  ameboid 
movement,  resembling  the  motion  of  the  leucocytes  of  the  blood  and  of 
true  amebae.     The  plasmic  contents  are  more  distinctly  granular  than 
that  of  the  leuco-sphaerocytes  and  the  vacuolesare  generally  fewer,  usually 
from  one  to  three.     They  resemble  amebae  excepting  that  there  is  no 
distinct  hyaloplasm  (ectoplasm).     In  the  ripe  tomato  these  bodies  are 
few  in  number  but  in  the  green  tomato  they  are  quite  numerous  and  show 
very  marked  ameboid  movement.     They  vary  from  15  to  30  microns  in 
diameter.     Most  of  them  contain  chlorophyll,  from  three  to  twenty  or 
even  more  unchanged  chlorophyll  granules  of  the  same  type  and  kind  as 
occur  in  the  normal  peripheral  pulp  cells  of  the  green  tomato.     They 
encyst  quite  readily  and  in  this  form  they  cannot  be  distinguished  from  the 
larger  leuco-sphaerocytes,  especially  those  which  are  free  from  chlorophyll 
granules.     Since  these  actively  motile  cells  occur  in  the  intact  fruit  tissues, 
especially  abundant  in  the  mucilaginous  layer  of  the  tomato  seed,  it  is 
reasonable  to  assume  that  they  are  derived  from  the  plasmic  elements  of 
the  tomato  itself  and  are  not  true  amebae  which  might  have  entered  from 
the  outside.     Perhaps  10  per  cent,  of  the  total  number  of  the  sphaerocytes 
are  of  the  ameboid  type  and  about  70  per  cent,  of  these  contain  chloro- 
phyll.   There  are  indications  that  all  of  the  sphserocytes  pass  through  the 
ameboid  stage. 

What  analogy  there  may  exist  between  the  amebo-sphaerocytes  of  the 
tomato  and  the  amebocy  tes  of  the  Spongilla  group  is  not  determined.  The 
sponge  amebocytes  which  are  said  to  arise  from  the  archeocytes  are  capa- 
ble of  ameboid  movement  and  will  form  into  new  sponge  cell  aggregates. 
That  the  amebo-sphaerocytes  of  the  tomato  are  capable  of  multiplying  by 
septation  is  probable  and  that  they  may  form  new  tissue  cell  aggregates 


134 


PHARMACEUTICAL  BACTERIOLOGY 


is  a  fact  if  the  postulate  herein  submitted,  to  the  effect  that  they  are  im- 
mature pulp  cells,  is  correct. 

c.  Nudeo-sphcerocytes. — These  resemble  the  leuco-sphaerocytes  as  to 
form  and  as  to  presence  of  vacuoles,  but  differ  in  that  they  contain  nuclei 
and  distinct  nucleoli  and  usually  also  brown  coloring  matter.  The  nuclei 
are  comparatively  large  and  resemble  those  of  the  mature  pulp  cells  of  the 
tomato.  Dull  brown  to  reddish  brown  chromophores  are  usually  aggre- 
gated about  the  nucleus.  There  is  no  evidence  of  the  presence  of  an 
outer  membrane  or  cell-wall.  In  the  green  tomato  the  nucleo-sphaero- 
cytes  may  also  show  chlorophyll  granules  aggregated  about  the  nucleus. 


FIG.  47. — Nucleo-sphaerocytes  from  hanging  drop,  twelve  days  old.  A,  living 
nucleated  sphaerocytes.  B,  dying  sphaerocytes  showing  extruding  vacuoles.  C, 
septation  (?)  as  observed  in  hanging  drop.  D,  a  group  ot  dead  sphasrocytes  in  hanging 
drop. 

The  most  remarkable  characteristic  of  the  nucleo-sphaerocytes  is  their 
great  vitality.  Crushed  tomato  pulp  in  the  hanging  drop  incubated  at 
25°  to  30°  C.  and  also  kept  at  normal  room  temperature,  showed  that  the 
nucleo-sphaerocytes  will  remain  alive  for  many  months  whereas  the  normal 
tissue  cells  and  inclusive  of  most  other  forms  of  sphaerocytes,  die  almost  at 
once  or  at  the  longest  within  a  period  of  twenty-four  to  forty-eight  hours. 

d.  Chromo-spharocytes. — These  are  generally  larger  than  the  nucleo- 
sphaerocytes  and  contain  a  variable  number  of  reddish  brown  coloring 
bodies  (chromophores),  irregular  in  outline,  apparently  identical  with 
the  coloring  bodies  of  the  tomato  parenchyma.  Abundant  vacuoles  are 
found.  In  the  hanging  drop,  the  chromophores  become  diffused  through 
the  plasmic  substance  of  the  sphaerocyte,  losing  their  morphological  iden- 
tity entirely. 


SYMBIOLOGY — THE   BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS      135 

e.  Chloro-sphcerocytes. — These  are  comparatively  few  in  the  ripe  to- 
mato.    In  the  unripe  tomato  they  are  very  abundant.     They  may  also 
contain  brown  chromophores  and  they  generally  have  distinct  nuclei. 
The  chlorophyll  granules  are  elliptical  in  form  and  are  identical  with^he 
chlorophyll  granules  of  the  peripheral  pulp  cells  of  the  tomato. 

Chlorophyll  granules  are  very  short  lived  outside  of  the  mother  cell. 
In  the  hanging  drop  they  disintegrate  very  readily,  much  more  readily 
than  do  the  brown  coloring  bodies. 

f.  Amylo-spharocytes. — These  are  always  nucleated  and  are  simply 
nearly  mature  sphaerocytes  which  contain  a  few  starch  granules.     They 
may  also  contain  brown  chromophores  arid  chlorophyll  granules. 


PIG.  48. — Sphaerocytes  of  the  grape.  A,  C,  Mature  pulp  cells.  B,  D,  sphaerocytes 
in  various  stages  of  deVolpment. 

Observations  in  the  hanging  drop  showed  that  the  chloro-sphaerocyte 
and  the  amylo-sphaerocyte  died  as  quickly  as  did  the  mature  pulp  cells. 
The  amyloplastids  are  however  quite  resistent  and  under  certain  condi- 
tions will  continue  growth  for  some  time  in  the  hanging  drop.  Asparagin 
(i-iooo)  appears  to  stimulate  the  amyloplastids. 

Hanging  drops  of  the  tomato  pulp  rich  in  sphaerocytes,  incubated  at 
3°°  to  35°  C.  showed  that  many  of  the  nucleo-sphaerocytes  kept  alive  for 
fifteen  months  and  longer,  whereas  most  other  forms,  including  the 
mature  tissue  cells,  usually  died  within  a  period  of  twenty-four  hours. 
The  most  marked  change  in  the  living  nucleo-sphaerocytes  in  the  hanging 
drop  was  a  slight  increase  in  size  of  the  cell  and  of  the  nucleus  and  a  very 
pronounced  darkening  of  the  plasmic  substance.  At  first  the  vacuoles 
increased  in  number  and  also  in  size,  some  of  the  more  peripheral  ones 


i36 


PHARMACEUTICAL  BACTERIOLOGY 


becoming  extruded.  In  the  course  of  four  or  five  weeks  the  vacuoles 
gradually  became  smaller  and  smaller  and  finally  disappeared  alto- 
gether. In  time  many  of  the  sphaerocytes  developed  plasmic  threads 
which  extended  from  side  to  side  across  the  cell.  In  many  cases  the  nucleus 
divided  into  from  several  to  perhaps  as  many  as  thirty  and  more,  second- 
ary nuclei;  these  secondary  nuclei  being  invariably  smaller  than  the 
mother  nucleus. 


PIG.  49. — Sphaerocytes  of  the  pulp  cells  of  the  immature  squash.  A,  pulp  cell; 
a.'nucleus  with  irregular  branching  nucleolus;  b,  sphaerocytes  in  various  stages  of  de- 
velopment; c,  starch  granules;  d,  plasmic  granules,  some  of  which  are  capable  of  very 
active  movement.  B,  nucleus  enlarged,  showing  irregular  and  branching  nucleolus  and 
outer  irregular  branching  particles.  C,  a  sphaerocyte  more  highly  magnified,  showing 
outer  irregular  branching  particles  resembling  those  of  the  nucleus.  D,  illustrating 
motion  of  two  plasmic  granules  (a,  b),  which  meet  at  (c),  where  they  come  to  rest.  E, 
illustrating  a  more  complex  form  of  motion  of  plasmic  granules. 

Fig.  49,  x  1000;  D  and  E  represent  the  directions  and  distances  traveled  by  four 
different  plasmic  granules  within  a  period  of  about  5  seconds,  going  at  a  speed  of  from 
3  to  8  microns  per  second.  At  that  rate  the  plasmic  granule  travels  from  0.18  to  0.48 
millimeters  in  one  minute. 

One  of  the  functions  of  the  sphaerocytes  is  to  continue  the  life  of  the 
plant  part,  as  fruit  or  seed,  after  the  plant  part  has  become  separated  from 
the  mother  plant.  That  is,  the  plasmic  activities  are  centered  in  the 
sphaerocytes,  rather  than  in  the  cell  plasm  or  in  the  nucleus  of  the  mother 
cell.  A  second  and  perhaps  equally  important  function  is  the  warding  off 
of  infections.  The  nucleo-sphaerocytes  in  particular  show  a  high  resist- 
ance to  the  successful  invasion  by  the  various  organisms  of  infection,  as 
rotting  bacteria,  yeasts  and  molds.  Hanging  drop  cultures  rich  in  sphae- 
rocytes remained  free  from  infection  even  with  rather  careless  manipula- 


SYMBIOLOGY — THE  BIOLOGICAL   RELATIONSHIPS   OF   ORGANISMS     137 

tion  and  occasional  exposure  to  air.  The  greater  keeping  power  of  ripe 
fruit,  as  compared  with  green  fruit  of  the  same  kind,  is  no  doubt  due  to 
the  fact  that  the  ripe  fruit  contains  comparatively  more  sphaerocytes 
than  does  the  green  fruit.  The  further  study  of  these  highly  interesting" 
structures  in  the  plant  kingdom,  and  their  analogues  in  the  animal  king- 
dom, will  no  doubt  reveal  new  chapters  in  the  study  of  cytology  and  also 
in  immunology. 

The  entire  discussion  of  cytology,  of  the  relationship  of  the  cells  (so- 
matic as  well  as  germatic)  of  living  organisms,  unicellular  as  well  multicel- 
lular,  etc.,  is  intimately  associated  with  the  problems  of  symbiosis  in  the 
broader  sense,  as  has  been  indicated  in  the  preceding.  We  shall  now  take 
up  the  more  specific  forms  of  symbioses,  generally  recognized  as  such. 

2.  The  Phenomena  of  Symbiosis 

Introduction. — All  living  organisms  manifest  a  more  or  less  intimate 
biological  interdependence  and  relationship.  In  fact,  their  very  existence 
depends  upon  this  condition;  therefore  no  organism,  no  matter  how 
simple  or  how  complex  its  structure  may  be,  is  the  result  of  a  wholly  inde- 
pendent phylogenetic  development.  Upon  careful  study  and  investiga- 
tion it  is  found  that,  although  this  interrelation  and  interdependence 
varies  greatly  as  to  quality  and  quantity,  there  may  be  found  innumerable 
intermediary  phenomena  which  make  it  difficult  to  draw  the  dividing  lines. 
Such  a  difficulty  is,  for  instance,  encountered  in  attempting  to  distinguish 
between  mere  "associations"  or  societies  (according  to  Warming  and 
others)  and  true  symbiosis.  Both  are  evident  phenomena  of  biological 
interdependence  with  the  general  difference  that  in  the  former  the  inter- 
dependence is  remote,  in  the  latter  more  close. 

Great  difficulty  is  encountered  in  limiting  and  defining  the  biological 
relationships  in  the  animal  kingdom.  Highly  automobile  organisms  do 
not  permit  the  ready  establishment  of  symbiotic  relationships  as  we  have 
come  to  understand  them.  Symbiosis  presupposes  a  certain  relative 
fixedness  of  the  organisms.  We  may  find  clearly  defined  symbioses  be- 
tween highly  automobile  organisms  and  those  which  are  comparatively 
non-motile.  Here  it  is  very  essential  to  keep  distinct  the  difference 
between  auto-mobility  and  passive  motility  (immobility).  The  former 
tends  to  counteract  or  reduce  the  occurrence  of  symbiosis;  the  latter 
favors  its  occurrence  as  well  as  its  adaptive  modification,  as  will  be  ex- 
plained later  in  the  discussion.  The  most  clearly  defined  and  most  highly 
specialized  forms  of  symbiosis  occur  between  non-motile  organisms. 

Motility  or  non-motility  of  organisms  has  little  or  no  direct  influence 
upon  the  more  remote  biological  relationships.  From  the  fact  that 
these  latter  phenomena  are  most  conveniently  limited,  geographically, 


138  PHARMACEUTICAL  BACTERIOLOGY 

it  becomes  evident  that  they  are  largely  dependent  upon  the  influence  of 
the  soil,  the  climate,  moisture,  etc.  (meteorological  influences). 

The  largest  and,  at  the  same  time,  the  most  remote  association  of 
organisms  is  the  hemispherical.  The  faunal  and  floral  differences  between 
the  eastern  and  western  hemispheres  are  considerable,  as  every  naturalist 
can  testify.  In  each  hemisphere  we  again  recognize  subdivisions  of 
associations,  which  may  be  designated  as  zonal.  Here  the  interdepen- 
dence is  more  marked,  and  is  primarily  dependent  upon  the  influence  of 
temperature  and  light.  The  fauna  and  flora  of  the  tropics  is  essentially 
different  from  that  of  the  temperate  zone,  and  this  again  is  different  from 
the  arctic.  Each  of  the  zonal  areas  is  again  subdivided  into  numerous 
larger  or  smaller  geographically  delimited  societies,  dependent  upon  local 
influences,  as  soil,  elevation,  moisture,  sunlight,  etc.  For  example,  life 
in  the  Mississippi  valley  is  essentially  different  from  that  in  the 
Rocky  mountain  region.  In  each  of  these  divisions  we  again  find  numer- 
ous smaller  socie  ties .  The  process  of  subdividing  could  be  carried  on  indefi- 
nitely. These  smaller  subdivisions  may  be  natural  or  artificial,  as  pond, 
brooklet,  meadow,  field,  roadside,  town,  city,  etc.,  each  of  which  has 
its  peculiar  fauna  and  flora. 

Within  each  of  these  numerous  associations,  great  and  small,  we  find 
the  organisms  acting  and  reacting  upon  each  other.  Here^there  seems  to 
be  a  mutualistic  association  of  two  or  more  organisms,  while  the  next  door 
neighbors  may  be  engaged  in  a  fierce  struggle  for  existence.  A  single 
example  will  suffice  to  illustrate  this.  The  wood-peckers  and  trees 
evidently  form  a  mutualistic  association,  while  insects  and  larvae  are 
diligently  hunted  by  the  wood-pecker.  Weasel  and  wood-pecker  again  are 
antagonistically  related. 

Definition  of  Symbiosis.. — Etymologically  the  word  symbiosis  signifies 
" a  living  together."  It  is  therefore  peculiarly  fitted  for  use  in  the  broader 
sense,  as  including  all  phenomena  of  "living  together."  Owing  to  the 
mutability  and  imperfections  of  a  language  the  etymology  of  a  word  is  not 
sufficient  to  limit  its  application.  A  careful  definition  or  explanation  is 
always  necessary.  Symbiosis  may  be  defined  as  a  contiguous  association 
of  two  or  more  morphologically  distinct  organisms,  not  of  the  same  kind, 
resulting  in  a  loss  or  acquisition  of  assimilated  food-substances.  This 
definition  is  by  no  means  perfect.  It  will,  however,  be  left  to  further  dis- 
cussions to  point  out  and  explain  its  deficiency. 

The  Origin  of  Symbiosis. — It  is  self-evident  that  before  a  symbiotic 
relationship  between  morphologically  distinct  organisms  could  be  estab- 
lished it  was  absolutely  necessary  that  they  be  brought  in  close  proximity, 
or  in  actual  contact.  It  is  also  clear,  from  a  priori  reasoning,  that  there 
could  be  no  inherent  tendency  within  these  organisms  to  attract  or  repel 


SYMBIOLOGY — THE   BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS     139 

each  other;  nor  could  the  first  contact  have  been  co-incident  with  morpho- 
logical and  physiological  adaptations.     The  very  conception  of  symbiosis 
implies  something  secondary,  and  in  a  certain  sense  something  abnormal.^ 
The  establishment  of  marked  symbit>ses  required  long  periods  of  time; 
just  when  they  began  is  impossible  to  determine.     It  is,  no  doubt,  justi- 
fiable to  assume  that  a  number  of  lowly  organized  organisms  existed  in  a 
natural  state,  manifesting  no  symbiotic  phenomena,  because  competition 
(for  space)  had  not  yet  resulted  from  over-production.     It  may  also  be 
assumed  that  symbiotic  phenomena  began  to  manifest  themselves  during 
the  earliest  geologic  ages.     All  the  multitudinous  phenomena  of  antagonis- 
tic symbiosis,  and  of  mutualistic  symbiosis,  represent  highly  specialized 
biological  conditions  which  were  initiated  by  the  first  contact  of  morpho- 
logically distinct  organisms.     This  contact  set  up  a  new  phase  in  the 
environment.     An  unforseen  struggle  was  the  result,  since  it  is  reasonable 
to  assume  that  the  primal  contact  relationships  of  contiguous  organisms 
was  antagonistic  rather  than  mutualistic.     As  already  indicated,  organ- 
isms were  not  primarily  adapted  to  form  or  enter  into  symbiotic  relation- 
ships, yet  there  is  every  reason  to  suppose  that  natural  tendency  must  have 
been  to  respond  toward  the  contact  organism  as  toward  the  environing  sub- 
strata.    The  living  organism  takes  its  nourishment  and  other  essentials 
for  existence,  from  the  environment.     The  organism  induces  destructive 
or  katabolic  changes  in  its  environment,  and  the  foreign  organism  with 
which  it  was  accidentally  brought  in  contact,  was  simply  a  new  bit  in  the 
environment.     This  primal  or  incipient  antagonism  engendered  by  this 
accidental  contact  of  living  organisms,  being  essentially  mutualistically 
antagonistic,  as  indicated,  must  have  tended  to  drive  the  contiguous  organ- 
isms away  from  each  other,  which  they  were  free  to  do,  for  as  yet  there  were 
no  morphological  adaptations  by  which  one  organism  might  attach  itself 
to  another.     Subsequently,  the  antagonism  may  have  been  increased  or 
even  entirely  modified,  changing  into  mutualism  or  other  highly  complex 
symbiotic  associations.     These  changes  are  intimately  bound  up  with  the 
questions  of  "struggle  for  existence,"  "survival  of  the  fittest,"  "evolution," 
"adaptive  changes,"  etc.,  etc.    We  may  cite  the  example  of  parasitic 
fungi  for  the  purpose  of  explaining  the  probable  origin  of  antagonistic 
symbiosis.     Most  fungi  are,  no  doubt,  derived  from  algae,  as  certain  mor- 
phological similarities  would  lead  us  to  believe.     Owing  to  lack  of  space, 
or  to  over-productiveness,  certain  algae  frequently  came  in  contact  with 
more  highly  organized  plants  and  animals,  from  which  they  absorbed 
(by  osmotic  action)  various  organic  food-substances,  thereby  reducing  the 
necessary  activity  of  chlorophyllian  assimilation.     Co-incident  with  the 
first  contact  and  resultant  change  in  function,  there  was  a  corresponding 
change  in  structure.     As  the  opportunities  for  the  symbiotic  association 


I4O  PHARMACEUTICAL  BACTERIOLOGY 

continued  (perhaps  more  or  less  interruptedly),  the  morpho-physiological 
changes  progressed  in  the  direction  of  parasitism  and  away  from  inde- 
pendence. Finally  the  originally  independent  chlorophyll-bearing  and 
carbon  assimilating  organism  became  wholly  dependent  upon  an  organic 
food  supply  and  sustained  a  total  loss  of  the  chlorophyllian  function. 
There  is  no  doubt  that  the  host  plant  or  host  plants  are  also  more  or  less 
affected  by  the  symbiosis.  The  relative  morpho-physiological  changes 
are  approximately  in  proportion  to  the  size  (volume)  and  biological  ac- 
tivity of  the  associating  organisms. 

It  is  necessary  to  keep  distinct  the  difference  between  mere  associations 
and  societies  of  organisms,  and  symbioses  proper.  There  is  scarcely  a 
problem  of  economic  significance  which  is  not  directly  associated  with 
some  form  of  symbiotic  relationship  of  organisms.  One  needs  but  call 
to  mind  the  recent  discoveries  in  the  treatment  of  disease,  modern  surgery, 
agriculture,  dairy  industries,  etc.  A  mere  mention  of  all  the  experimenta- 
tions and  discoveries  in  connection  with  symbiosis  would  fill  volumes. 
Much  careful  research  is  yet  necessary  in  order  to  clear  up  the  uncertain- 
ties in  regard  to  the  biological  significance  of  many  of  the  symbioses.  In 
order  to  impress  this  uncertainty  more  fully  we  shall  mention  a  few  symbi- 
otic phenomena  which  are  either  not  recognized  as  such,  or  improperly 
classified,  usually  as  parasitism. 

Unclassified  Symbiotic  Phenomena. — Under  this  heading  will  be 
briefly  mentioned  numerous  and  varied  phenomena  which  are  of  undoubted 
symbiotic  nature,  but  are  not  as  yet  clearly  understood.  Some  of  these 
phenomena  are  of  a  very  complicated  nature  and  indicate  a  long  phylo- 
genetic  development.  In  many  instances  the  morphological  adaptation 
and  relationship  of  the  organisms  is  so  remote  as  to  awaken  serious  doubt 
as  to  its  symbiotic  nature.  Under  this  category  belong  the  mutual 
adaptation  of  plants  (entomophilous  and  other  flowers)  and  insects;  also 
the  various  forms  of  mimicry;  the  association  of  various  species  of  aphidae 
and  ants  upon  certain  plants ;  besides  many  other  phenomena.  The  associa- 
tion of  trees,  such  as  the  myrmocophilous  Cecropias  and  representatives  of 
other  genera,  with  ants,  is  by  many  designated  as  true  mutualistic  symbiosis. 

The  relation  of  the  male  and  female  reproductive  cells  is  of  a  truly 
symbiotic  nature.  It  represents  a  highly  specialized  form  of  individual- 
ism. The  relationship  existing  between  the  immature  embryo  and  the 
food-supplying  parent-stock  is  evidently  a  form  of  symbiosis.  There  are 
numerous  instances  in  both  the  animal  and  vegetable  kingdom  in  which 
the  more  or  less  imperfect  but  complete  second  generation  lives  in  a  sym- 
biotic relationship  with  the  first  generation.  The  relationship  existing 
between  sporophytic  and  gametophytic  generations  may  be  considered 
symbiotic  in  nature  even  though  the  two  generations  are  parts  of  the 


SYMBIOLOGY — THE   BIOLOGICAL    RELATIONSHIPS    OF    ORGANISMS     141 

same  ontogeny.  The  two  generations  form  a  highly  specialized  symbio- 
sis (individualism).  There  are  many  other  phenomena  of  a  compli- 
cated nature  which  are  designated  as  true  parasitism  by  some  authors 
while  others  do  not  ascribe  to  them  any  symbiotic  relationship. 

Several  species  of  crab  belonging  to  the  genus  Stenorhynchus  are  usually 
covered  by  a  growth  of  algae,  sponges  and  other  lower  plants  and  animals. 
This  is  perhaps  a  case  of  accidental  symbiosis.  The  habitat  of  the  crab 
combined  with  its  slow  movement  makes  the  chitinous  skeleton  a  suitable 
substratum  for  the  attachment  of  various  aquatic  organisms.  The  cover- 
ing may  serve  some  protection  but  this  is  evidently  of  no  significant 
importance.  Species  of  the  closely  related  genus  Inachus  are  also  covered 
by  a  similar  growth  but  here  the  plants  and  animals  serve  as  food  for  the 
crab.  Brehm  states  that  the  crab  even  transplants  hydroids,  algae  and 
other  organisms  upon  its  back,  thus  converting  itself  into  a  traveling 
economic  zoologic  and  botanic  garden.  Another  crab  is  totally  hidden 
by  sponges  growing  upon  it  which  enables  it  to  approach  its  prey  un- 
perceived  as  well  as  to  hide  from  its  enemies.  Although  some  of  these 
phenomena  seem  very  complicated,  there  is  no  evidence  of  marked  sym- 
biotism.  If  more  than  mere  accidental  symbiotism  does  exist,  no  experi- 
ments have  been  made  to  demonstrate  whether  it  is  antagonistic  or 
mutualistic. 

The  hermit  crab  is  morphologically  adapted  to  live  in  the  empty  shells 
of  certain  snails.  The  last  pair  of  legs  are  much  shortened  and  serve  the 
special  function  of  holding  the  shell.  The  coleopter  Necrophilus  subter- 
raneous attacks  live  snails,  eats  the  animal  and  then  moves  into  the  empty 
shell.  The  crayfish  Phronima  sedentaria  eats  species  of  Doliolum  and 
Pyrosoma  and  utilizes  the  empty  skeleton  as  a  dwelling  place,  paddling 
it  about  by  means  of  its  claws.  Although  these  phenomena  are  in  part  of 
symbiotic  nature,  yet  one  must  hesitate  to  place  them  in  this  category 
since  the  hunting,  killing  and  eating  process  is  not  true  parasitism)  antago- 
nistic symbiosis).  According  to  definition,  symbiosis  necessitates  a  pro- 
longed contiguous  relationship.  This  is  not  the  case  with  the  carnivorous 
animals  and  their  prey.  The  apparently  wonderful  adaptations  of  the 
crab  and  other  related  animals,  to  the  snail-shell  and  to  the  outer  skeletons 
of  crustaceans,  is  perhaps  purely  accidental  unless  it  can  be  proven  that 
the  structural  conformations  are  the  result  of  phylogenetic  development. 

Climbing  plants  are  interesting  as  they  mark  the  beginnings  of  a  highly 
complicated  form  of  symbiosis.  The  plants  form  a  close  association  with 
their  supports,  which  in  most  cases  are  living  plants;  especially  is  this  the 
case  in  the  dense  jungles  of  the  tropics.  Whether  these  plants  cling  to 
their  support  by  means  of  twining  stems,  tendrils,  suctorial  organis  or 
aerial  roots,  there  is  more  or  less  absorption  of  soluble  food-substances 


142  PHARMACEUTICAL  BACTERIOLOGY 

from  the  living  support  and  in  so  far  it  constitutes  a  symbiotic  relationship. 
The  morphological  adaptations  favoring  climbing  are  however  primarily 
for  the  purpose  of  bringing  the  assimilative  tissues  nearer  the  sunlight, 
and  away  from  excessive  moisture.  The  support  is  necessary  in  order 
to  enable  them  to  enter  into  successful  competition  with  other  plants. 
In  many  instances  the  supporting  plant  plays  the  part  of  a  host  as  in 
true  parasitism  (Cuscuta).  There  is  little  doubt  that  the  members  of 
the  Dodder  family  were  originally  climbing  plants  which  took  almost 
their  entire  nourishment  from  the  soil  and  air.  The  contact  with  the 
supporting  plants  gradually  developed  a  wholly  parasitic  habit.  In 
many  of  the  climbing  plants  the  supporting  function  predominates  while 
the  symbiotic  relationship  remains  practically  zero.  This  is  especially 
true  of  the  large  thick-stemmed  climbers  of  the  tropics. 

Highly  interesting  though  little  understood,  are  the  frequently  occur- 
ring neoformations  in  animals,  such  as  tumors  (epithelioma,  limpoma, 
osteoma,  sarcoma,  carcinoma,  etc.)  and  cysts  of  various  kinds.  It  is 
supposed  that  these  growths  are  neoformations  arising  from  the  develop- 
ment of  dormant  embryonic  cells.  These  pathologic  growths  are  special 
body  cell  proliferations,  as  has  already  been  stated.  Why  certain  tissues 
should  suddenly  take  on  this  highly  antagonistic  attitude  toward  the 
rest  of  the  body  cells  (somatic  cells)  is  not  known.  It  is  a  fact  that  these 
circumscribed  degenerative  cell  proliferations  present  all  of  the  charac- 
teristics of  a  foreign  attacking  parasite,  sapping  the  vitality  and  even 
destroying  the  life  of  the  host.  Much  attention  has  been  given  to  cancer 
research  within  recent  years  but  no  conclusions  have  as  yet  been  reached, 
neither  as  to  cause  or  as  to  cure. 

In  conclusion  we  shall  cite  a  few  symbioid  phenomena  from  the  insect 
world  and  show  how  they  are  gradually  converted  into  undoubted  sym- 
bioses.  Different  species  of  wasps  narcotize  or  paralyze  spiders,  crickets 
and  caterpillars,  by  stinging,  thus  rendering  them  motionless.  How  the 
wasp  learned  to  perform  this  remarkably  delicate  operation,  through  which 
the  animal  operated  upon  is  paralyzed  and  rendered  entirely  helpless 
without  destroying  life,  is  not  known.  Not  even  the  most  skilled  surgeon 
now  living  can  perform  an  operation  of  this  kind  with  the  precision  and 
the  nicety  with  which  the  apparently  awkward  and  plundering  wasp  per- 
forms it.  In  this  condition  the  narcotized  insects  are  sealed  into  the 
wasp's  nest  containing  the  eggs,  in  order  to  serve  as  food  for  the  young 
wasp.  This  condition  becomes  more  complicated  by  the  intrusion  of 
another  wasp  which  unobserved  lays  its  egg  in  the  nest  already  supplied 
with  the  necessary  food.  The  foreign  egg  develops  first  and  the  young 
wasp  not  only  eats  the  food  supplied  by  its  foster  mother,  but  also  the 
eggs.  From  these  conditions  to  true  parasitism  is  only  a  step.  Some 


SYMBIOLOGY — THE  BIOLOGICAL   RELATIONSHIPS    OF   ORGANISMS     143 

wasps  lay  their  eggs  directly  into  the  tissues  of  the  caterpillar.  The 
egg  develops  and  the  young  larva  feeds  upon  the  less  vital  tissues  of  the 
host  so  as  to  prolong  life  as  much  as  possible.  Finally  only  the  outer 
tegument  of  the  host  remains  which  is  utilized  as  a  protective  covering 
during  the  resting  stage. 

We  must  also  mention  the  phenomena  of  grafting  (plant),  tissue  trans- 
plantation, tumor  transplantation,  gland  transplantation,  organ  transplan- 
tation, etc.  These  are  usually  not  designated  as  symbioses.  In  successful 
tree  grafting,  for  example,  there  is  established  an  apparently  perfect  sym- 
biosis of  a  mutualistic  character.  In  successful  cancer  transplantation 
there  is  established  a  form  of  symbiosis  which  is  truly  parasitic.  Skin 
grafting,  as  practised  in  cases  of  severe  burns  constitutes  a  symbioid  cell 
association  of  the  mutualistic  type. 

Phenomena  of  True  Symbiosis. — The  phenomena  of  symbiosis  here 
denned  have  been  more  or  less  discussed  by  scientists  and  have  received 
recognition.  Authors  are,  however,  at  variance  as  to  their  exact  limita- 
tions which  makes  the  definitions  subjectively  variable.  The  phenomena 
of  symbiosis  may  be  classified  as  follows : 

I.  Incipient  Symbiosis  (Indifferent  Symbiosis). 

1.  Accidental  Symbiosis. 

2.  Contingent  Symbiosis  (Raumparasitismus) . 
II.  Antagonistic  Symbiosis. 

1.  Mutual  Antagonistic  Symbiosis  (Mutual  Parasitism). 

2.  Antagonistic  Symbiosis  (Parasitism). 

a.  Obligative  Antagonistic  Symbiosis. 

b.  Facultative  Antagonistic  Symbiosis. 

3.  Saprophytism. 

a.  Facultative  Saprophytism. 

b.  Obligative  Saprophytism. 
III.  Mutualistic  Symbiosis. 

1.  Nutricism  (Semi-mutualistic  Symbiosis). 

2.  Mutualism. 

3.  Individualism. 

a.  Semi-individualism. 

b.  Complete  Individualism. 

IV.  Compound  Symbiosis. 

V.  Cytosis. 

1.  Autocytosis. 

a.  Patrocytosis — Phagocytosis,  tissue  regeneration,  etc. 

b.  Paracytosis — Epithelioma,  cysts,  etc. 

2.  Heterocytosis.     Consortism.     Commensalism. 


144  PHARMACEUTICAL   BACTERIOLOGY 

These  phenomena  are  represented  by  the  association  of  widely,  differ- 
ent organisms.  Organisms  similar  to  those  which  enter  into  an  antago- 
nistic symbiosis  may  also  occur  in  mutualistic  symbiosis.  This  seems 
to  indicate  that  the  development  of  these  associations  depends  largely 
upon  opportunity  (environment).  To  some  extent,  however,  the  organ- 
isms control  or  modify  the  symbiotic  relationship.  A  classification  of 
the  phenomena  indicating  their  phylogenetic  relationship  can  therefore 
not  be  based  upon  the  phylogenesis  of  the  organisms  which  enter  into 
heir  formation.  One  can  only  indicate  the  physiological  relationship 
of  the  phenomena  and  their  approximate  relative  evolution. 

i    Incipient  Symbiosis  (Indifferent  Symbiosis) 

Under  incipient  symbiosis  are  included  the  multitudinous  phenomena  of 
symbiotic  relationships,  which  have  not  yet  acquired  evident  antagonistic 
or  mutualistic  characters.  In  many  instances  there  are  marked  morpho- 
logical adaptations,  but  without  any  apparent  corresponding  functional 
modification.  In  far  the  greater  number  of  cases  there  is  simple  contact, 
resulting  from  over  production.  In  view  of  this  fact  one  may  be  criticised 
for  recognizing  such  relationships  as  symbioses.  From  a  priori  reasoning 
one  is,  however,  forced  to  conclude  that  the  first  symbiotic  activities  began 
with  the  first  contact  of  organisms.  Incipient  symbiosis,  therefore,  forms 
the  basis  or  common  source  of  all  symbiotic  phenomena.  From  it  gradu- 
ally emerged  highly  complicated  morphological  and  physiological  adapta- 
tions of  originally  independent  (self-sustaining)  organisms.  There  is  also 
little  doubt  that  as  our  methods  of  investigation  become  more  highly 
perfected,  many  of  the  symbiotic  phenomena  now  considered  as  indifferent 
will  be  relegated  to  the  realms  of  antagonistic  or  mutualistic  symbiosis. 

i.  Accidental  Symbiosis. — This  represents  the  least  specialized  form  of 
symbiosis,  but  is  of  wider  occurrence  than  all  the  others  combined.  Acci- 
dental symbiosis  is  represented  by  the  mere  coming  in  contact  of  two  or 
more  morphologically  distinct  organisms;  such  contact  being,  however, 
sufficiently  prolonged  to  give  it  the  semblance  of  a  symbiosis.  Mere 
momentary  contact  is  not  symbiosis  as  here  understood. 

Accidental  symbioses  are  particularly  numerous  where  there  is  luxuri- 
ant growth,  hence  where  competition  is  great,  as  in  the  tropics  and  in 
highly  productive  soils  generally.  The  lower  parts  of  plants  in  green-houses 
are  covered  with  bacteria,  hyphal  fungi,  algae  and  more  rarely  some  of  the 
lower  protozoa.  The  epidermal  cells  of  many  plants  contain  more  or  less 
bacteria.  Submerged  plants  are  covered  with  mollusks,  hydras,  tubulari- 
ians,  amebas,  vorticellas,  etc.  The  larger  land  and  water  organisms 
furnish  hiding  places  and  protection  for  hosts  of  smaller  organisms. 


SYMBIOLOGY — THE  BIOLOGICAL  RELATIONSHIPS   OF   ORGANISMS     145 

In  fact,  no  organism  is  free  from  the  accidental  association  with  other 
organisms. 

In  all  of  the  instances  mentioned  there  is  no  perceptible  evidence  of 
either  antagonism  or  mutualism.  Injurious  results  may  result,  due  to 
mechanical  causes.  Slight  morphological  changes  usually  result,  but  such 
changes  seem  to  have  no  effect  upon  the  life-history  and  development  of 
the  symbionts. 

To  the  category  of  accidental  symbiosis  also  belong  the  association  of 
climbing  plants  and  their  living  supports.  The  symbiotic  relationship  was 
at  first  merely  accidental.  It  is  a  striking  example  illustrating  how  marked 
and  highly  specialized  morphological  adaptations  favoring  one  function 
may  initiate  widely  different  morpho-physiological  changes.  In  the  case 
of  climbing  plants,  it  is  impossible  to  know  when  the  symbiotic  relation- 
ship begins  to  overbalance  the  function  of  mechanical  support.  It  is  just 
as  difficult  to  determine  when  marked  symbiotic  phenomena  began  to 
manifest  themselves.  It  is  safe  to  conclude,  however,  that  the  morpho- 
logical changes  favoring  climbing  and  support  progressed  considerably 
before  any  marked  symbiotic  relationships  occurred. 

It  is  also  evident  that  accidental  symbiosis  is  a  condition  readily  sub- 
ject to  change,  since  the  permanency  of  symbioses  is  in  direct  proportion 
to  the  degree  of  mutualistic  specialization.  Each  plant  and  animal  may 
enter  into  accidental  symbiotism  with  other  plants  and  animals.  In  a 
given  animal  this  association  changes  with  a  change  of  locality,  of  tem- 
perature, or  of  moisture;  in  fact,  with  every  change  in  the  environment. 
The  absence  of  all  permanency  in  morphological  and  functional  relation- 
ship, characterizes  accidental  symbiosis.  It  resembles  a  form  of  hap- 
hazard experimentation  on  the  part  of  nature  to  determine  whether  or 
not  a  definite  symbiotic  relationship  can  be  established. 

2.  Contingent  Symbiosis. — In  this  form  of  symbiosis  the  relationship 
of  the  organisms  is  already  sufficiently  marked  to  give  the  semblance  of  an 
elective  affinity,  although  the  functional  interdependence  is  as  yet  not 
manifest.  It  is  of  wide  occurrence  among  widely  different  organisms. 
Many  phenomena  heretofore  recognized  or  variously  classified  as  parasit- 
ism, perhaps  belong  to  this  category.  Most  of  the  phenomena  recog- 
nized by  the  German  scientists  as  Raumparasitismus  also  belong  here. 
The  citation  of  a  few  examples  will  suffice  to  explain  the  nature  of  contin- 
gent symbiosis,  and  to  distinguish  it  from  mere  accidental  symbiosis  as 
well  as  from  the  more  highly  specialized  forms  of  symbiosis. 

There  is  a  difference  between  the  bacterial  flora  of  the  digestive  tract 
of  man  and  that  of  the  chicken  or  dog.     Certain  bacteria,  which  have  not 
yet  become  markedly  antagonistic  or  mutualistic  in  their  symbiotic  as- 
sociations", show  a  preference  for  one  digestive  tract  which  indicates  that 
10 


146  PHARMACEUTICAL  BACTERIOLOGY 

there  must  be  some  elective  affinity.  That  the  elective  affinity  is  only 
slight  is  evident  from  the  fact  that  the  bacteria  referred  to  will  very  readily 
grow  and  multiply  upon  artificial  culture  media,  and  may  be  induced  to 
change  hosts.  Some  algae  show  an  elective  affinity  for  certain  living 
substrata.  Sirosiphon  puhinatus  occurs  quite  constantly  upon  species  of 
Umbilicaria  and  Gyrophora.  Pleuro coccus  punctiformis  occurs  upon  the 
young  thallus  of  Cladonia  and  Baeomyces.  Pleurococcus  vulgaris,  on  the 
other  hand,  occurs  upon  the  most  varied  substrata  living  and  dead;  hence 
this  association  is  evidently  only  accidental,  as  the  alga  shows  no  preference 
for  any  particular  host.  It  has,  perhaps,  a  slight  biologic  preference  for 
some  of  the  Polyporei. 

Some  of  the  higher  crustaceans  select  certain  corals,  among  which  they 
live,  without  forming  any  marked  symbiotic  relationship.  In  one  locality 
(geographical  area)  Hydra  mridis  seems  to  prefer  one  vegetable  substra- 
tum (Nuphar),  while  in  another  locailty  it  prefers  to  live  upon  another 
plant,  Lemna  polyrhiza.  Some  Rotifera  show  a  preference  for  certain 
plants  to  which  they  attach  themselves.  Certain  algae,  as  species  of 
Dactylococcus  and  Euglena,  show  a  decided  tendency  to  locate  upon  such 
animals  as  cyclops,  snails,  and  clams.  Some  mammals  (sloth,  ant-eater, 
and  others),  have  algae  living  upon  them.  The  symbiosis  of  snails  with 
corals  is  perhaps  contingent.  Some  sponges  and  hydroids  show  a  prefer- 
ence for  animals,  others  for  plants.  Marine  life  in  particular,  presents 
many  forms  of  contingent  symbioses.  The  instances  cited  are  sufficient 
to  indicate  the  nature  of  contingent  symbiosis.  Many  require  further 
careful  study  before  anything  definite  can  be  stated  as  to  their  biological 
activity  and  as  to  their  relationship  to  other  symbioses. 

II.  Antagonistic  Symbiosis 

The  phenomena  included  under  this  head  are  of  wide  occurrence  and 
were  the  first  to  receive  the  attention  of  scientists.  The  term  as  here  used 
includes  mutual  antagonistic  symbiosis  and  antagonistic  symbiosis  proper. 
The  former  is  not  generally  recognized  by  authors.  The  latter  is  more 
commonly  known  as  parasitism.  There  are  no  objections  to  the  use  of  the 
term  parasitism,  since  it  has  become  clearly  defined  and  definitely  restricted 
in  its  application.  It  is,  however,  recommended  that  the  term  antagonis- 
tic symbiosis  be  substituted  for  the  sake  of  uniformity  in  terminology. 

From  the  nature  of  things  the  morpho-physiological  specializations  and 
adaptations  of  antagonistic  symbiosis  are  limited.  Although  one  of  the 
symbionts  maybe  highly  benefitted  the  other  is  always  injuriously  affected. 
This  injurious  effect  may  finally  reach  the  stage  where  it  will  react  upon 
the  parasite,  thus  indirectly  resulting  in  the  mutual  destruction  of  the 
symbionts.  In  far  the  greater  number  of  instances  the  host  is  not  de- 


SYMBIOLOGY — THE  BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS     147 

stroyed,  nor  even  seriously  injured,  although  its  morphological  changes 
tend  in  that  direction;  a  condition  which  will  of  necessity  react  upon  the 
parasite.  From  this  it  also  becomes  evident  that  it  is  desirable  for  the 
parasite  to  locate  upon  a  host  whose  vitality  and  biological  activities-are 
many  times  greater  than  its  own.  This  we  find  to  be  the  case,  the  host 
is  quite  generally  a  large  plant,  while  its  parasites  are  comparatively 
small. 

Strictly  speaking,  antagonistic  symbiosis  is  therefore  a  destructive 
association.  The  morphological  and  physiological  changes  tend  toward 
dissolution  rather  than  evolution.  It  is  a  change  from  the  higher  to  the 
lower,  hence  a  katabolic  change.  There  is,  however,  no  doubt  that  sym- 
bioses  which  were  originally  antagonistic  may  subsequently  be  converted 
into  mutualistic  symbiosis.  Reinke  expresses  the  opinion  that  the  lichen 
prototype  was  the  result  of  the  parasitic  association  of  a  fungus  and  an 
alga(Nostoc).  This  transition  from  antagonism  to  mutualism,  however, 
takes  place  early  in  the  phylogeny  of  the  symbiosis. 

As  has  already  been  indicated,  the  majority  of  symbioses  were  perhaps 
originally  more  or  less  antagonistic,  although  actual  experiments  are 
wanting  to  prove  this.  Incipient  antagonistic  symbioses  are,  however,  in 
existence,  represented  by  some  Chlorophyceae  and  Cyanophyceae,in  and 
upon  higher  plants.  In  time  these  algae  will  no  doubt  lose  their  chloro- 
phyllian  function  and  depend  entirely  upon  the  organic  food  supply  of 
the  host. 

i.  Mutual  Antagonistic  Symbiosis  (Mutual  Parasitism). — Mutual 
parasitism  as  such  has  heretofore  received  little  or  no  recognition. 
It  is  a  phenomenon  characterized  by  the  mutual  antagonism  of  the 
symbionts  and  is  therefore  essentially  different  from  antagonistic 
symbiosis  proper,  or  parasitism.  It  is  a  relationship  which  can  not 
readily  occur.  If,  for  example,  two  or  more  symbionts  nearly  equal 
in  size  and  in  vitality,  enter  into  a  relationship  of  mutual  antagonism 
two  things  may  occur.  Owing  to  the  antagonism  a  prolonged  sym- 
biosis is  impossible,  and  the  symbionts  will  return  to  the  original 
substrata,  or  they  will  mutually  destroy  each  other.  It  is,  however, 
highly  probable  that  an  association  of  organisms,  which  was  at  first 
more  or  less  mutually  antagonistic,  later  developed  into  antagonistic 
symbiosis  proper  or  even  into  mutualistic  symbiosis.  As  an  illustra- 
tion of  mutual  antagonistic  symbiosis,  we  may  mention  the  cells  of 
any  pathologic  growth,  as  carcinoma,  epithelioma,  etc.  The  cells  com- 
posing the  growth  are  antagonistic  toward  each  other,  as  well  as  toward 
the  normal  cells  of  the  organism  upon  which  they  occur.  This  antago- 
nistic relationship  of  the  tumor  forming  cells  is  indicated  by  the  fact  that 
they  are  much  weakened  in  comparison  with  the  normal  body  cells.  In 


148  PHARMACEUTICAL  BACTERIOLOGY 

i 

the  numerous  cases  of  socalled  tissue  degeneration  the  cells  of  the  partic- 
ular tissue  assume  a  dystrophic  relationship  toward  each  other,  resulting 
in  their  mutual  destruction,  carrying  with  them  the  host  which  gave  them 
origin  and  of  which  they  formed  a  part. 

Complete  and  simultaneous  mutual  antagonism  of  symbionts  of  equal 
potentiality  or  virulency  is  certainly  of  rare  occurrence.  Further  careful 
study  may  reveal  phenomena  of  this  nature.  Various  forms  of  mutual 
antagonism  do,  however,  occur.  It  exists,  for  example,  between  normal 
cells  of  plants  and  animals  and  certain  disease-producing  germs  (bacteria, 
etc.)  The  ability  of  the  cells  to  resist  the  attacks  of  certain  germs  is 
spoken  of  as  "physiological  resistance"  or  "natural  resistance."  In 
fact,  the  recent  investigations  and  discoveries  in  regard  to  immunity 
are  based  upon  this  mutual  antagonism  between  host  and  parasite. 
This  antagonism  varies  greatly  between  different  organisms.  Phagocyto- 
sis is  another  example  of  mutual  antagonism.  Under  ordinary  circum- 
stances the  phagocytes  destroy  all  of  the  germs  with  which  they  come  in 
contact,  thus  preventing  the  occurrence  of  diseases  or  other  intoxication. 
Under  certain  conditions  the  germs,  however,  gain  the  upper  hand  and 
destroy  the  phagocytes. 

2.  Antagonistic  Symbiosis  (Parasitism). — Antagonistic  symbiosis  in 
some  of  its  forms  is  familiar  to  all.  We  will  briefly  mention  some  of  the 
more  important  relationships  of  host  and  parasite. 

In  many  instances  the  host  is  destroyed  without  any  preliminary 
morphological  changes.  The  parasite  simply  enters  the  cells  and  destroys 
them  by  assimilating  the  plasmic  contents.  This  form  of  symbiosis 
Tubeuf  designates  as  Perniciasm.  In  other  instances,  also  belonging  to 
perniciasm,  there  are  slight  secondary  changes  morphologically  before 
death  takes  place,  as  galls  or  swellings. 

In  other  instances  death  is  the  result  of  enzymatic  action,  or  due  to 
ptomaines  or  toxins  produced  by  the  parasite,  as  in  various  diseases  of 
animals  as  well  as  of  plants.  Some  parasites  dissolve  the  cell- wells  of  the 
host,  while  others  simply  lie  in  contact  with  the  cells  and  absorb  the 
contents  by  osmotic  action.  In  a  great  number  of  instances  hyper- 
trophies and  abnormalities  in  growth  are  induced  (galls,  hypertrophied 
fruits  and  leaves;  enlargements  in  animal  tissues).  Again,  atrophy,  or  a 
total  check  in  development,  may  occur. 

In  some  forms  of  parasitism  the  host  adaptation  has  become  highly 
specialized.  In  the  phenomena  known  as  heteroecism  the  successive 
generations  of  the  parasite  develop  upon  different  host-plants.  For 
example,  Puccinia  graminis  developes  its  aecidiospores  upon  Berberis 
vulgaris,  while  its  teleutospores  are  developed  upon  some  of  the  grasses, 
as  wheat  or  oats.  Most  parasites  do,  however,  not  have  successive  auto- 


SYMBIOLOGY — THE   BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS      149 

genetic  generations.     Many  are  limited  to  one  host-species,  or  even  to 
definite  tissues  or  organs. 

One  organism  may  enter  into  different  forms  of  symbiosis.  For 
example,  the  bacillus  of  typhoid  fever  may  enter  into  an  accidental' 
(perhaps  contingent)  symbiosis  with  the  oyster,  while  with  man  it  forms 
an  antagonistic  symbiosis.  The  bacillus  of  Asiatic  cholera,  likewise,  may 
live  in  and  upon  various  animals  without  any  injurious  effects,  but  as  soon 
as  it  finds  its  way  to  the  intestinal  canal  of  man  it  acts  as  a  true  parasite. 
The  differentiation  into  facultative  and  obligative  parasitism  depends 
upon  the  ability  that  some  organisms  have  of  living  as  parasites  and  sapro- 
phytes, while  others  are  absolutely  dependent  for  their  existence  upon 
association  with  the  living  host. 

The  most  common  parasites  are  the  fungi.  The  Schizomycetes  form 
antagonistic  symbioses,  preferably  with  animals.  The  higher  parasitic 
fungi  predominate  upon  vegetable  tissue.  Many  diseases  of  animals 
are  also  due  to  the  higher  fungi.  Algae  occur  parasitically  in  and  upon 
plants  and  animals.  Many  of  the  Chlorophyceae  and  Cyanophyceae 
occur  as  parasites  upon  higher  plants.  Many  of  the  marine  algae  are 
parasitic  upon  each  other  as  well  as  upon  marine  animals.  Higher  plants 
are  often  parasitic  (Mistletoe,  Dodder,  Indian  Pipe,  etc.).  Protozoa  occur 
parasitically  upon  animals.  A  sporozoan  causes  malaria.  Still  higher 
animals  occur  parasitically  in  and  upon  animals  and  plants,  producing 
manifold  injurious  effects. 

Most  interesting  is  the  phenomenon  of  sex-parasitism  in  which  one 
sex,  usually  the  male,  lives  parasitically  upon  the  other.  In  one  of  the 
parasitic  crustaceans  the  male  is  entirely  dependent  upon  the  female  for 
its  sustenance.  Among  the  Bouellias  the  male  is  represented  by  a  mere 
fertilizing  structure,  parasitic  within  the  reproductive  organs  of  the  female. 

We  may  also  mention  the  parasitic  relationship  of  embryos  and  the 
mother-organisms.  This  has  already  been  referred  to  as  a  questionable 
form  of  symbiosis.  Klebs  is,  however,  of  the  opinion  that  it  is  true  para- 
sitism. The  embryo  of  a  plant  derives  all  its  nourishment  from  its  parent, 
and  in  addition  takes  from  it  certain  materials  which  it  stores  for  future 
use  (cotyledons,  endosperm).  Even  after  birth  the  young  of  many  ani- 
mals remain  in  parasitic  association  with  the  parent.  Of  the  numerous 
eggs  of  the  black  salamander,  only  one  develops  a  young  animal,  which 
eats  the  remaining  eggs. 

3.  Saprophytism. — Saprophytism  is  not  true  symbiosis.  This  is  a 
condition  which  in  many  instances  was  no  doubt  phylogenetically  de- 
rived from  parasitism  as  we  have  all  gradations  between  obligative  para- 
sites and  obligative  saprophytes.  It  is  quite  reasonable  to  assume  that  in 
many  cases  of  parasitism  in  which  the  death  of  the  host  was  the  final 


150  PHARMACEUTICAL  BACTERIOLOGY 

outcome,  the  parasite  adapted  itself  to  the  dead  substance  and  used  it  as 
food.  In  some  instances  saprophytism  no  doubt  originated  as  such. 
Dead  organic  matter  occurs  plentifully  everywhere  and  forms  a  suitable 
substratum  for  a  number  of  animal  as  well  as  vegetable  organisms,  having 
special  morpho-physiological  adaptations  for  utilizing  such  as  food  supply. 
This  preference  was  no  doubt  gradually  acquired. 

III.     Mutualistic  Symbiosis 

This  form  of  symbiosis  differs  from  the  preceding  in  that  the  relation- 
ship of  the  organisms  is  mutually  beneficial.  Each  symbiont  possesses 
or  has  developed  a  specific  character  which  is  useful  for  the  other  symbionts. 
As  in  the  preceding  forms  of  symbioses,  widely  different  organisms  may 
enter  into  its  formation.  The  morphological  changes  accompanying  the 
functional  adaptations  may  be  very  marked  or  scarcely  perceptible,  nor 
is  the  adaptation  quantitatively  and  qualitatively  equal  for  all  the  sym- 
bionts. The  adaptation  is  complementary,  one  organism  supplies  a 
deficiency  (morphological  or  physiological)  of  the  others.  Theoretically 
there  is  no  limit  to  the  degree  of  specialization  and  perfection  which  this 
form  of  symbiosis  may  attain.  In  fact,  mutualistic  symbiosis  implies 
that  there  is  an  increased  specialization  and  fitness  to  enter  into  the  struggle 
for  existence.  This  is  most  beautifully  illustrated  in  the  case  of  lichens. 
These  plants  are  of  wider  distribution  and  possess  greater  vitality  and 
physiological  activity  than  either  of  the  symbionts.  They  occur  in  the 
tropics  as  well  as  in  the  extreme  north;  in  the  lowest  valleys  as  well  as  on 
the  highest  mountain  peaks.  Bonnier  has  shown  that  their  vitality  is 
greater  than  that  of  any  other  morphologically  similar  plants.  Likewise 
the  -mutualistic  symbiosis  occurring  in  the  Leguminosae,  adapts  these 
almost  equally  well  to  rich  and  poor  soil,  thus  giving  them  a  great  advan- 
tage over  other  plants.  Our  knowledge  of  the  higher  forms  of  mutualistic 
symbiosis  is  as  yet.  too  incomplete  to  permit  us  to  make  statements  as  to 
the  full  benefits  resulting  therefrom. 

i.  Nutricism. — Nutricism  establishes  a  connecting  link  between  the 
lesser  marked  symbioses  and  mutualism.  It  may  be  defined  as  a  form  of 
symbiosis  in  which  one  symbiont  nourishes  the  second  symbiont  without 
receiving  any  benefit  in  return.  It  might  therefore  be  designated  as  one 
sided  or  incomplete  mutualism.  Absolute  nutricism,  as  above  defined, 
does  perhaps  not  occur,  for,  as  already  indicated,  it  is  not  reasonable  to 
assume  that  any  symbiotic  relationship  exists  in  which  all  of  the  sym- 
bionts are  not  more  or  less  mutually  affected.  There  are,  however,  a  few 
instances  in  which  one  symbiont  is  very  materially  benefited,  while  the 
other  is  not  materially  benefited.  The  most  marked  example  is  met 
with  in  the  mycorhiza  of  the  Cupulif  erae.  A  mycorhiza  is  the  association 


SYMBIOLOGY — THE   BIOLOGICAL   RELATIONSHIPS    OF    ORGANISMS     151 

of  a  hyphal  fungus  with  the  younger  rootlets.  The  function  of  the  fun- 
gus, which  forms  a  network  about  the  tips  of  the  terminal  rootlet,  is  to 
supply  the  tree  with  certain  food-substances  and  moisture  taken  from  the 
soil.  It  also  supplants  the  function  of  the  hair-cells  which  are  wanting  in 
the  mycorhiza.  It  has  been  proved,  experimentally,  that  the  tree  is  greatly 
benefited,  while  no  evidence  could  be  found  to  indicate  that  the  fungus  is 
benefited.  The  hyphae  always  remain  on  the  outside  of  the  root,  and 
therefore  form  an  ectotrophic  association.  The  endotrophic  mycorrhiza 
of  orchids  have  not  yet  been  sufficiently  studied  to  determine  the 
kind  of  symbiosis  which  they  represent.  Tubeuf  designates  it  as 
nutricism. 

In  Cycas  revoluta  we  find  a  form  of  symbiosis  which  is  evidently  nutri- 
cism. It  is  found  that  in  the  majority  of  cultivated  cycads  there  are 
numerous  tubercular  outgrowths  from  the  roots,  which  usually  contain  a 
species  of  Nostoc  between  the  cells  of  a  specialized  parenchyma.  This  is 
evidently  not  a  form  of  parasitism  as  is  indicated  by  the  fact  that  the 
cycads  bearing  the  greater  number  of  tubercles  are  in  no  wise  injuriously 
affected;  neither  has  it  been  proven  that  the  host  is  benefited.  There  is, 
however,  no  doubt  that  the  Nostoc  is  dependent  upon  the  host  for  its  food 
supply.  It  may  therefore  be  looked  upon  as  a  case  of  nutricism,  in  which 
the  host  acts  as  the  transfer  agent. 

Klebs  cites  an  interesting  example  which  is,  no  doubt,  nutricism.  The 
crayfish  Pagurus  Prideauxii  is  quite  uniformly  infested  by  one  of  the 
actinias  (Adamsia  palliata).  The  latter  is  said  to  be  absolutely  dependent 
upon  the  former  for  its  food-supply.  The  crayfish  receives  only  a  slight 
benefit  if  any. 

2.  Mutualism. — This  form  of  symbiosis  was  first  described  byReinke 
and  de  Bary  among  botanists  and  van  Beneden  and  Klebs  among  zoolo- 
gists. By  mutualism  is  meant  a  form  of  symbiosis  in  which  the  symbionts 
mutually  benefit  each  other,  but  are  still  capable  of  leading  an  independ- 
ent existence.  It  is  an  association  of  wide  occurrence  and  in  many 
instances  reaches  a  high  degree  of  morphological  and  physiological 
specialization. 

The  most  striking  example  occurs  in  the  root-tubercles  of  the  Legum- 
inosae.  The  tubercles  are  neoformations  induced  by  the  Rhizobia  which 
grow  and  multiply  in  the  parenchyma  cells.  The  Rhizobia  take  their  food 
supply  .direct  from  the  plasmic  and  other  cell  contents  of  the  host;  in 
return  the  latter  receives  certain  nitrogenous  compounds  formed  by  the 
bacteria  in  the  process  of  binding  the  free  nitrogen  of  the  air.  It  has  been 
proven  experimentally  that  the  symbionts  may  exist  independently,  but 
thrive  much  better  when  in  association,  especially  in  poor  soil. 

To  this  category  also  belong  the  association  of  ants  and  trees  in  the 


152  PHARMACEUTICAL  BACTERIOLOGY 

tropics,  which  has  already  been  referred  to.  A  given  species  of  ant  lives 
upon  and  obtains  its  food  supply  from  the  branches  of  a  tree  (Cecropia) ; 
in  return  the  ants  protect  the  tree  against  the  attacks  of  another  species 
of  ants.  The  ants  live  within  the  transversely  divided  hollow  stem  to 
which  they  gain  access  by  eating  away  the  thin  lateral  (outer)  area. 
The  thin  outer  membrane  of  which  there  is  one  to  each  hollow  chamber 
and  the  chambers  themselves  are,  however,  perhaps  not  the  result  of  the 
symbiotic  association.  The.  preexisting  morphological  characters  simply 
happen  to  make  possible  the  establishment  of  this  particular  form  of 
symbiosis. 

In  the  insectivorous  plants  (Drosera,  Dionaea,  Nepenthes)  we  doubtless 
have  another  example  of  mutualism.  Formerly  it  was  generally  believed 
that  the  plant  itself  digested  the  insects  which  it  caught,  by  the  aid  of 
irritable  glandular  hairs  and  other  special  organs.  It  is  highly  probable 
that  the  insect  digesting  ferment  is  secreted  by  bacteria  which  live  upon 
the  plant. 

A  most  remarkable  instance  of  mutualism  occurs  in  the  animal  king- 
dom. The  very  inactive  polyp  Actinia  prehensa  lives  firmly  attached  to 
the  inner  sides  of  the  claws  of  the  crustacean  Melia  tessellata.  The 
Actinia  aids  in  killing  the  prey  of  the  crayfish  while  the  latter  carries  its 
guest  from  place  to  place  thus  giving  it  better  opportunities  for  securing  a 
sufficient  food-supply.  Mobius  states  that  this  association  occurs  with 
all  the  representatives  of  Melia  tessellata,  both  male  and  female,  and 
that  it  is  almost  impossible  to  remove  the  symbionts  without  injuring 
them. 

Many  of  the  symbiotic  associations  of  algae  with  animals  are  perhaps 
mutualistic.  Many  Actinias  contain  single-celled  algae  which  elaborate 
food-substances  for  the  use  of  the  polyp.  Brandt  states  that  as  long  as 
this  animal  contains  no  algae,  it  feeds  upon  the  organic  substances  in  the 
immediate  vicinity,  but  as  soon  as  it  becomes  associated  with  the  algae  it 
depends  upon  these  for  the  supply  of  organic  food.  Further  research  is 
necessary  to  determine  whether  or  not  this  is  true  mutualism. 

3.  Individualism. — This  form  of  symbiosis  differs  from  mutualism  in 
that  one  or  more  of  the  symbionts  is  absolutely  dependent  upon  the  other 
for  its  existence.  It  therefore  represents  a  more  highly  specialized  form 
of  mutualism,  from  which  it  is  no  doubt  phylogenetically  derived. 
Individualism  may  be  divided  into  semi-individualism  and  complete 
individualism.  In  the  former  at  least  one  of  the  symbionts  is  incapable  of 
existing  independently,  however,  the  organism  of  which  it  was  a  part  can- 
not survive.  In  complete  individualism  none  of  the  several  symbionts 
can  continue  to  exist  independently.  A  new  individual,  a  new  autonomy, 
is  the  result.  It  cannot  be  denied  that  the  association  of  the  symbionts 


SYMBIOLOGY — THE  BIOLOGICAL   RELATIONSHIPS   OF   ORGANISMS     153 

is  less  close  and  even  less  interdependent  than  it  is  among  the  several 
living  inclusions  of  the  cell  of  higher  plants  and  animals,  and  even  less 
closely  interdependent  than  the  associations  of  the  somatic  cells  of  the 
higher  multicellular  organisms,  but  is  an  independent  autonomous  struc= 
ture  nevertheless. 

(a)  Semi-individualism. — This  is  perhaps  of  wide  occurrence.  It  is 
represented  by  the  lower  lichens  in  which  the  algal  symbiont  is  capable  of 
leading  an  independent  existence,  while  the  fungus  can  not.  In  the  lowest 
crustaceous  lichens  there  is  perhaps  mere  mutualism,  since  several  investi- 
gators state  that  the  symbionts  may  live  independently  as  fungus  and 
alga.  Some  algae  seem  to  form  semi-individualism  with  animals.  Ac- 
cording to  Kiihn,  Pleurococcus  brachypodis  and  Pleurococcus  chlopodis  occur 
only  upon  the  body  (among  the  hair)  of  the  two  and  three-toes  sloths. 
Simple-celled,  chlorophyll-bearing  algae  or  chlorophyll-bodies  have  been 
found  in  representatives  of  the  following  genera  of  the  animal  kingdom; 
Ameba,  Dactylospora,  Difflugia,  Hyalosphsenia,  Helepera,  Arcella, 
Cochliopodium,  Actinosphaerium,  Rhaphidiophrys,  Acanthocystis,  Het- 
erophrys,  Chondropus,  Spaerastrum,  Ciliophrys,  Vorticella,  Epistylis, 
Ophrydium,  Vaginicola,  Euplotes,  Urostyla,  Uroleptus,  Stichotricha, 
Spirostomum,  Blepharisma,  Climacostomum,  Stentor,  Cyrtostomum, 
Micro  thorax,  Paramecium,Loxodes,  Coleps,Lionotus,  Amphileptus,Lacry- 
maria,  Phyalina,  Holophrya,  Euchelyodon,  Euchelys,  Spongilla,  Hydra, 
Vortex,  Mesostomum,  Hypostomum,  Derostomum,  Couroluta,  Anthea, 
Bouellia,  Idotea.  In  many  instances  the  green  particles  occurring  within 
the  animals  are  simply  remnants  of  chlorophyll  derived  from  the  algae 
upon  which  the  animal  feeds.  In  other  instances  there  is  an  undoubted 
symbiotic  association  of  an  alga  and  the  animal. 

(6)  Complete  Individualism. — The  best  known  and  perhaps  the  most 
typical  form  of  complete  individualism  is  represented  by  the  higher  lichens. 
Most  authors  are  agreed  that  the  fungal  symbiont  has  entirely  lost  the 
power  of  independent  existence,  while  the  alga  may  exist  independently. 
Some  recent  experiments  would,  however,  indicate  that  the  algae  likewise 
have  lost  the  power  of  continued  independent  existence.  Lichens  would 
therefore  form  a  complete  individualism.  The  association  of  algae  with 
Hydra  viridis  and  with  forms  of  soil  amebae  and  ciliata,  perhaps 
belongs  to  this  category. 

The  true  significance  of  the  lichen  symbiosis  does  not  receive  general 
recognition.  Botanists  still  persist  in  classifying  them  among  the  fungi. 
Some  place  them  in  a  separate  and  independent  group,  recognizing  the 
fact  that  they  "are  not  as  other  plants"  but  steadfastly  refuse  to  recognize 
the  true  reason  why  they  should  be  given  an  independent  group  position. 
Most  of  the  botanists  see  in  the  relationship  between  alga  (the  gonidia  of  the 


154  PHARMACEUTICAL  BACTERIOLOGY 

older  lichenologists)  and  the  fungus  which  make  up  the  lichen  individual, 
nothing  more  or  less  than  parasitism.  Some  consider  the  fungus  as  the 
parasite,  others  the  alga.  Fiinfstiick,  in  his  grouping  of  the  lichens  in 
Engler  and  Prantl's  Pflanzenfamilien,  indeed  gives  them  a  place  of  their 
own  but  nevertheless  designates  them  as  fungi  parasitically  associated 
with  algae.  This  is  all  the  more  remarkable  since  Fiinfstiick  very  con- 
cisely sets  forth  those  morphological,  physiological  and  chemical  char- 
acteristics of  lichens,  which  clearly  indicate  their  autonomous  nature. 
He  refuses  to  look  upon  the  relationship  of  fungus  and  alga  as  mutually 
beneficial,  and  designates  it  as  a  special  or  peculiar  form  of  parasitism 
("Eine  besondere  Art  von  Parasitismus ") .  It  is  furthermore  a  mis- 
apprehension of  the  expression  "mutualistic  symbiosis"  to  interpret  it  as 
meaning  that  the  several  symbionts  are  equally  benefited.  The  term 
simply  implies  that  the  several  symbiotic  components  are  benefited 
(which  is  frankly  admitted  by  Funfstiick)  but  that  one  may  receive  the 
greater  return  favor  or  benefit.  There  are  some  botanists  who  refuse  to 
recognize  in  this  wonderful  biological  relationship  anything  more  than 
ordinary  parasitism.  Such  a  deduction  is  possible  only  when  the  compo- 
nents or  symbionts  are  considered  separately  and  not  in  their  mutual 
relationship.  For  example,  in  like  manner  it  is  possible  to  reach  the  conclu- 
sion that  the  domestic  animal  is  injuriously  affected  through  the  influence 
of  man,  or  that  civilized  rnan  himself  is  merely  a  parasitized  or  degenerate 
form  of  the  ignorant  savage.  To  speak  of  the  algal  (gonidial)  symbiont  as 
imprisoned  and  parasitized  is  as  irrational  as  to  speak  of  the  imprisoned 
and  parasitized  horse  or  cow.  It  is  very  true,  man  uses  the  milk,  the  hide, 
the  hair,  the  teeth,  the  meat,  the  bones,  the  hoof,  in  fact  every  part  of 
the  animal.  It  does  look  like  a  clear  case  of  the  most  pronounced  one- 
sided parasitism,  but  the  aspect  is  changed  markedly  as  soon  as  we  consider 
both  animals,  the  cow  and  the  man,  in  their  mutual  relationship.  Had 
it  not  been  for  man,  the  cow  would  perhaps  not  exist  at  all;  as  it  is,  millions 
of  these  animals  enjoy  a  life  of  luxury  as  compared  with  the  life  they  would 
be  compelled  to  lead  as  independent  unparasitized  wild  animals.  Who 
can  then  say  that  the  relationship  is  not  mutualistic?  By  analogy  the 
same  argument  applies  to  the  alga  and  fungus  in  the  lichen-group,  only 
here  we  have  a  true  symbiotic  relationship.  While  it  is  generally  admitted 
that  the  lichen  components  or  symbionts  may  develop  and  exist  in- 
dependently under  artificial  conditions,  at  least  up  to  a  certain  stage  of 
development,  there  is  no  evidence  that  such  is  the  case  in  nature.  The 
statement  has  been  made  that  the  algal  symbiont  may  escape  from  the 
thallus  and  vegetate  independently  on  bark,  etc.,  but  it  lacks  proof. 
Even  though  that  were  the  case,  the  fungal  symbiont  does  not  exist 
independently  in  nature  and  hence  a  lichen  is  an  impossibility  without 


SYMBIOLOGY — THE  BIOLOGICAL  RELATIONSHIPS    OF   ORGANISMS     155 

the  mutualistic  association  of  alga  and  fungus.  No  one  has  yet  succeeded 
in  forming  a  lichen  by  associating  a  true  alga  (Cystococcus)  with  a  true 
ascomycetous  fungus.  If  this  were  possible  we  might  reasonably  excepL 
spontaneously  synthetic  lichen  formations  in  nature,  which  is  certainly 
not  the  case.  Lichens  invariably  arise  from  preexisting  lichens.  Some 
authorities  state  that  a  fungus  may  attack  Nostoc  colonies  and  transform 
them  into  collematous  lichens  but  this  statement  requires  verification. 

Therefore,  it  would  appear  that  the  most  plausible  and  reasonable 
attitude  to  take  toward  lichen  classification  is  to  consider  them  as  a 
distinct  class.  This  is  the  conclusion  reached  after  a  careful  study  of  the 
morphology  (gross  and  minute)  and  ecology  of  the  more  important 
representatives  of  this  very  interesting  group  of  plants. 

Future  experiments  may  demonstrate  that  the  living  inclusions  of 
the  cell  constitute  a  symbiotic  association  of  what  were  once  independent 
organisms  which  entered  into  a  mutualistic  association  which  has  now 
become  so  highly  specialized  that  we  fail  to  recognize  their  ancestral 
relationships  and  origin.  As  already  stated,  we  have  not  been  able  to 
induce  any  of  these  living  cell  inclusions  to  continue  existence  outside 
of  the  living  cell  of  which  they  are  a  part.  Some  years  ago  Reinke  ex- 
pressed the  opinion  that  some  skilled  scientist  of  the  near  future  would 
succeed  in  cultivating  chlorophyll  bodies  in  artificial  media.  We  know 
that  Carel  and  others  have  succeeded  in  inducing  tissue  proliferation  in 
artificial  media. 

IV.  Compound  Symbiosis 

By  compound  symbiosis  is  meant  the  association  of  two  or  more 
different  types  or  forms  of  symbioses.  Thus  we  may  have  two  or  more 
organisms  mutualistically  associated  with  each  other,  but  forming  a  com- 
mon antagonism  with  the  host.  It  has  long  been  recognized  that  most 
of  the  infections  are  not  simple,  but  rather  multiple.  In  tuberculosis 
of  the  lungs,  the  bacillus  of  tuberculosis  is  not  by  any  means  the  only 
infecting  organism  which  is  present,  other  bacteria  are  present,  also 
yeasts  and  molds.  The  leprosy  bacillus  will  not  thrive  unless  associated 
with  certain  symbionts,  as  amebae,  body  cells,  and  leprous  substance 
or  tissue.  The  Boas-Oppier  bacillus  is  a  fairly  constant  associate  with 
cancerous  tissue  and  this  organism  is  therefore  antagonistic  to  the  cancer, 
and  both  are  commensal  upon  the  body  of  the  cancerous  patient.  In 
Monostomum  bijugum,  a  parasitic  worm  found  in  birds,  it  is  known  that 
two  individuals  always  occur  together.  Most  abscesses  contain  from 
several  to  many  common  infecting  organisms.  The  Staphylococcus 
group  acting  as  the  pioneers,  preparing  the  way  for  the  entrance  of  the 
other  organisms,  which  feed  upon  the  products  resulting  from  the  primary 
infection. 


156  PHARMACEUTICAL  BACTERIOLOGY 

V.  Cytosis 

By  cytosis  is  meant  certain  cell  activities  which  partake  of  the  char- 
acter of  symbioses.  They  may  be  classed  into  autocytoses  and  hetero- 
cytoses.  In  the  former  cells  derived  from  and  forming  a  part  of  the 
organism  enter  into  cytotic  change.  In  the  second  instance,  the  cytotic 
activity  takes  place  in  cells  derived  from  some  other  organism.  The 
autocytose  may  be  divided  into  patrocytosis  and  paracytosis,  as  follows. 

Patrocytosis. — By  patrocytosis  is  meant  an  increased  activity  of  certain 
body  cells  for  the  purpose  of  protective  immunization  and  warding  off 
the  invasion  by  pathogenic  organisms.  The  best  example  is  phagocytosis, 
which  will  be  more  fully  explained  elsewhere.  Even  more  typical  are 
the  special  cell  activities  concerned  in  the  formation  of  healing  tissues  and 
in  regenerative  growths  of  all  kinds,  in  plants  and  in  animals.  Among 
the  higher  animals,  man  in  particular,  the  leucocytes,  the  lymphocytes, 
the  endothelial  and  epithelial  cells,  are  chiefly  engaged  in  the  patrocytotic 
activities.  In  plants  the  sphaerooytes  which  are  most  abundant  in  ripe 
fruits,  form  a  most  important  patrocytosis  (sphaerocytosis),  these  struc- 
tures being  thrown  off  from  the  cytoplasm  for  the  purpose  of  continuing 
the  life  of  the  plant  part  after  such  part  has  become  separated  from  the 
mother  plant.  They  also  ward  off  the  invasions  by  the  organisms  of 
decomposition.  The  sphaerocytes  do  not  begin  to  develop  abundantly 
until  active  cell  proliferation  (in  cambial  zone  and  apical  areas  and  else- 
where) has  ceased.  These  structures  evidently  continue  the  life  of  the 
cell,  after  cell  division  has  ceased., 

In  patrocytosis,  certain  body  cells,  that  is  cells  which  are  normal  to 
the  multicellular  organism,  perform  a  beneficient  relationship  with  the 
organism  of  which  they  are  a  part,  which  beneficient  relationship  is  in  every 
way  mutually  helpful.  The  patrocytes  occupy  a  subordinate  position 
toward  the  organism,  but  their  work  is  to  assist  in  the  maintenance  of  the 
state  of  health.  In  a  way,  they  bear  the  same  relationship  to  the  organism 
that  children  of  a  family  bear  to  the  head  of  the  family  in  a  well  regulated 
family.  The  patrocytes  take  their  origin  in  the  organism  and  they  are  abso- 
lutely dependent  upon  the  living  organisms  for  their  existence. 

Paracytosis. — This  is  the  opposite  of  patrocytosis.  In  paracytosis 
certain  body  cells  assume  an  antagonistic  relationship  toward  the  organ- 
ism of  which  they  are  a  part  and  from  which  they  took  their  origin.  Typ- 
ical examples  are  malignant  growths  of  all  kinds,  such  as  epithelioma, 
sarcoma,  carcinoma.  Here  again  the  epithelial  and  endothelial  cells  play 
an  important  part.  In  epithelioma  we  find  an  enormously  augmented 
pathologic  proliferation  of  epithelial  cells.  For  some  cause  or  causes 
yet  unknown  the  epithelial  cells  refuse  to  bear  a  normal  relationship  to 
the  rest  of  the  body  cells.  They  appear  to  have  become  vicious  strikers. 


SYMBIOLOGY — THE  BIOLOGICAL   RELATIONSHIPS    OF   ORGANISMS    157 

They  have  become  the  degenerates  and  perverts  among  the  body  cells. 
Instead  of  bearing  a  beneficient  relationship  toward  the  cells  which  gave 
them  origin,  they  do  all  within  their  power  to  destroy  the  cells  with  which^ 
they  are  associated. 

Occasionally  the  leucocytes,  the  lymphocytes  and  the  endothelial 
cells  of  the  capillaries  go  on  a  strike,  not  only  refusing  to  continue  the  per- 
formance of  their  normal  functions,  but  undergoing  active  disintegration 
thus  bringing  about  a  symptom  complex  which  soon  leads  to  the  death  of 
the  entire  organism,  as  in  purpura  hemorrhagica  and  in  pernicious  anemia. 
These]degenerative  changes  appear  to  have  some  intimate  interrelationship 
with  the  sympathetic  nerve  system.  It  would  appear  that  the  con- 
tinuous and  prolonged  suppression  of  the  emotional  feelings  and  sympa- 
thies leads  to  the  degenerative  cell  changes  mentioned.  No  doubt  the 
endocrine  secretions  are  also  profoundly  altered  in  these  cases.  Numer- 
ous fatalities  due  to  cellular  disintegrations  have  occurred  during  the  World 
War  among  those  who  for  various  reasons  were  obliged  to  completely 
suppress  or  hide  their  true  emotional  feelings.  The  peculiar  patho- 
genic cell  proliferation  encountered  in  malignant  growths  are  not  induced 
by  the  suppression  of  the  emotions.  Neither  the  direct  nor  the  inciting 
causes  of  these  formations  are  as  yet  known. 

By  heterocytosis  is  meant  a  foreign  cell  proliferation  in  or  upon  an 
organism.  Thus  cancer  tissue  may  be  transplanted  upon  a  mouse. 
Tissues  and  organs  may  be  transplanted  into  widely  distinct  animals. 
These  interesting  cell  proliferations  are  sufficiently  common  as  not  tor 
require  further  explanation  as  to  their  nature.  They  are  unquestionably 
of  symbiotic  nature. 


CHAPTER  VIII 
BACTERIA  IN  THE  INDUSTRIES.    UTILITARIAN  BACTERIOLOGY 

Because  of  the  fact  that  pathogenic  bacteria  received  the  major  atten- 
tion at  the  beginning  of  modern  bacteriological  study,  the  general  opinion 
gained  credence  that  all  bacteria  were  harmful  or  objectionable  in  some 
way.  This  is  far  from  the  actual  fact.  The  useful  bacteria  by  far  exceed 
the  harmful  kinds.  Not  only  is  this  true,  but  many  of  the  harmful  forms 
are  converted  into  various  beneficial  uses.  The  useful  bacteria  are  alto- 
gether too  much  neglected  by  the  student  of  bacteriology.  In  a  book 
of  such  limited  scope  it  is  not  possible  to  mention  all  of  the  uses  to  which 
bacteria  are  put  in  the  industries,  or  the  ultilitarian  part  they  play  in  hu- 
man economy.  The  following  discussion  is  intended  to  indicate  the 
importance  of  the  various  microorganisms  in  some  of  the  human  activities, 

I.  Bacteria  in  Agriculture 

Introduction. — Without  bacteria  the  higher  plants  and  animals  could 
not  exist.  As  is  known  the  carnivorous  animals  (meat  eating)  seize 
upon  herbivorous  animals  (plant  eating)  as  their  food  supply  and  the 
herbivora  feed  upon  the  higher  plants  which  in  turn  obtain  their  nourish- 
ment from  the  soil.  Now,  soil  is  nothing  more  nor  less  than  a  mixture 
of  dead  organic  matter,  bacteria,  sand  particles,  certain  chemical  com- 
pounds, with  a  variable  amount  of  moisture.  The  dead  organic  matter, 
commonly  called  humus,  is  derived  from  decomposed  plants  and  animals 
upon  which  the  soil  bacteria  feed,  in  the  presence  of  air  (oxygen),  warmth 
and  moisture.  The  sand  particles  are  derived  from  disintegrating  rock 
(the  result  of  bacterial  activity,  weathering,  and  water  erosion  effects), 
and  the  chemical  compounds,  so  essential  to  plant  life,  are  derived  from 
water  solutions  and  through  bacterial  activity. 

It  is  general  knowledge  that  as  soon  as  a  plant  or  animal  dies,  it  is 
at  once  decomposed  by  the  so-called  rotting  bacteria.  This  is  the  ultimate 
end  of  all  living  things.  These  decomposed  plants  and  animals  become 
mixed  with  the  soil  and  add  to  its  fertility  or  productiveness.  It  is  also 
general  knowledge,  based  upon  daily  observation,  that  organic  matter 
which  is  freely  exposed  to  air  and  warmth,  and  in  the  presence  of  moisture, 
undergoes  bacterial  decomposition.  There  are  indeed  many  conditions 
which  modify  these  bacterial  activities,  such  as  temperature,  air  supply, 

158 


BACTERIA   IN   THE   INDUSTRIES  159 

moisture  and  sunlight.  Most  bacteria  require  an  ample  supply  of  air 
(free  oxygen)  and  these  are  spoken  of  as  aerobes.  A  comparatively  smaller 
number  thrive  in  the  absence  of  air  (free  oxygen)  and  these  are  called 
anaerobes.  The  soil  bacteria  are  essentially  aerobes,  as  is  perhaps 
self-evident. 

Bacteria  are  however  not  the  only  microorganisms  found  in  the  soil. 
Soil  also  contains  minute  algae,  fungi  and  microscopic  single-celled  ani- 
mals (protozoa),  to  say  nothing  of  earthworms,  insects  and  other  higher 
animals,  concerned  in  certain  soil  changes  of  minor  importance.  The  pro- 
tozoa, algae  and  fungi  (molds)  may  be  and  are,  of  great  significance  in 
crop  growing.  In  a  general  way  it  may  be  stated  that  most  of  the  algae, 
the  fungi  and  the  protozoa  work  antagonistically  to  the  bacteria  which 
are  normal  to  soils.  It  must  however  not  be  supposed  that  all  of  the 
bacteria  found  in  the  soil  are  useful  or  beneficient.  Occasionally  harmful 
bacteria  develop  in  certain  soils,  causing  plant  diseases  and  otherwise 
interfering  with  plant  growth.  It  is  the  aim  of  modern  scientific  agri- 
culture to  so  regulate  cultural  operations  as  to  encourage  the  optimum 
development  of  the  beneficient  soil  bacteria,  reducing  at  the  same  time,  to  a 
minimum,  the  development  of  all  harmful  soil  organisms  (bacteria,  proto- 
zoa, molds,  etc.). 

Incidentally  it  may  be  mentioned  that  soil  harbors  bacteria  and  other 
organisms  which  play  no  part  in  plant  growth  or  in  agriculture  proper; 
such  as  the  lock  jaw  bacillus  (Bacillus  tetani),  the  anthrax  bacillus  (B. 
anthracis),  the  bacillus  of  malignant  oedema  (B.  Welchii),  the  tuberculosis 
bacillus  (B.  tuberculosis),  the  typhoid  bacillus  (B.  typhosis),  and  others. 
Soils  may  also  harbor  the  cause  of  rabies,  of  foot  and  mouth  disease,  the 
larvae  of  hook  worm  and  the  larvae  of  other  intestinal  parasites,  besides  a 
host  of  minute  organisms  injurious  to  plants  and  to  the  lower  animals. 
Swamp  lands  are  the  breeding  places  of  the  malaria  and  yellow  fever  carry- 
ing mosquitoes.  Proper  drainage  and  tillage  of  soils  tends  to  check  and 
to  reduce  to  a  minimum  the  development  of  these  highly  objectionable 
soil  inhabitants  and  thus  the  farmer  becomes  a  most  useful  worker  in 
behalf  of  public  health  and  sanitation.  Proper  sewage  disposal  prevents 
dysenteries  and  typhoid,  cholera  and  many  other  dread  diseases.  Every 
farmer  should  have  a  good  general  knowledge  of  rural  sanitation. 

Historical. — Crops  have  been  grown  for  thousands  of  years.  The 
earliest  cultural  methods  were  crude  indeed  and  at  that  remote  period 
nothing  was  known  about  soil  bacteriology,  but  even  in  Virgil's  time 
(about  70  B.C.,  hence  nearly  2,000  years  ago)  agriculture  had  made  some 
notable  progress,  for  the  noted  poet  in  his  Georgics,  clearly  sets  forth  the 
value  and  importance  of  turning  over  the  soil,  the  beneficial  results  of 
crop  rotation  and  the  value  of  vetch  and  of  other  leguminous  crop  plants 


l6o  PHARMACEUTICAL  BACTERIOLOGY 

for  enriching  the  soil.  Incidentally,  it  is  of  interest  to  know  that  Virgil's 
agricultural  epic  just  referred  to  was  primarily  intended  as  a  strong  plea 
for  turning  the  minds  of  the  idle  rich  back  to  the  soil,  a  plea  which  was 
never  more  urgent  than  it  is  at  the  present  time. 

The  Roman  farmers  practiced  crop  rotation,  summer  fallowing,  green 
manureing,  and  considered  the  bean,  the  vetch  and  luzerne  (alfalfa)  as 
special  enrichers  of  the  soil.  The  farmers  of  middle  Europe  early  acquired 
a  knowledge  of  these  cultural  operations  from  their  Roman  neighbors. 
Red  clover  soon  became  known  as  a  valuable  enricher  of  the  soil,  and  has 
been  long  used  for  that  purpose  by  the  farmers  of  France,  Germany  and 
England.  The  Chinese  and  Hindoos  have  for  thousands  of  years  made 
use  of  a  pressed  bean  fertilizer  in  rice  culture.  Transfer  of  a  rich  soil  top 
dressing  to  new  or  arid  fields  has  been  practiced  for  many  centuries.  In 
those  remote  times  the  value  of  soil  tillage,  of  soil  warmth,  of  fertilizers, 
was  fully  recognized  but  it  is  only  within  recent  years  that  the  true  signifi- 
cance of  these  basic  agricultural  factors  has  been  discovered. 

Plant  Growth  and  Bacteria. — The  bacteria  concerned  in  plant  growth 
may  conveniently  be  divided  into  three  great  groups,  as  follows: 

1.  Those  that  are  a  part  of  the  soil.     That  is,  those  bacteria  which  are 
normally  present  in  the  soil  and  which  feed  upon  the  organic  matter 
(humus)  in  the  soil,  rendering  the  humus  available  for  the  use  of  plants 
which  may  be  growing  in  the  soils. 

2.  Bacteria  which  live  upon  and  in  the  immediate  vicinity  of  the  roots 
of  plants.     These  bacteria  are  essential  to  the  normal  development  of 
plants  and  each  kind  of  plant  has  its  own  special  kind  or  kinds  of  bacterial 
associates.     The  bacteria  and  the  host  plants  form  a  mutualistic  associa- 
tion, that  is  an  association  for  mutual  gain  and  benefit  (mutualism,  or 
mutualistic  symbiosis). 

Organisms  other  than  bacteria  may  form  such  beneficient  associations 
with  higher  plants.  Thus,  we  find  molds  in  mutualistic  association  upon 
the  terminal  rootlets  of  oak  seedlings  and  upon  the  roots  of  other  repre- 
sentatives of  the  oak  family.  Some  of  the  soil  algae,  under  certain  condi- 
tions, will  enter  into  beneficient  relationships  with  certain  plants  (mints, 
calamus,  iris,  sedges,  and  other  wet  soil  plants).  An  alga  (Nostoc)  forms 
a  mutualistic  association  with  the  cycad  (Cycas  rewluta). 

3.  Bacteria  which  live  within  the  root  tissues  of  plants.     Perhaps  every 
farmer  and  gardener  has  observed  certain  nodules  on  the  roots  and  rootlets 
of  plants  belonging  to  the  bean  family,  as  bean,  pea,  lentil,  soy  bean, 
alfalfa,  clover,  peanut,  cassia,  lupine,  melilotus,  etc.     The  internal  tissue 
cells  of  these  nodules  contain  billions  of  bacteria  which  have  the  power  of 
binding  the  free  nitrogen  of  the  air,  converting  it  into  nitrogenous  com- 
pounds which  are  utilized  by  the  host  plants  and  which  upon  the  death  of 


BACTERIA   IN    THE    INDUSTRIES  l6l 

the  plants  reenter  the  soil,  enriching  it.  Pure  cultures  of  these  bacteria 
have  been  in  use  for  some  time  (over  thirty  years)  for  the  purpose  of 
increasing  the  crop  yield  in  virgin  soils  and  in  soils  deficient  in  nitro- 
genous compounds.  These  socalled  vest  pocket  fertilizers  are  extensively 
manufactured  in  the  United  States,  in  Canada  and  in  other  agricultural 
countries,  some  of  them  bearing  special  fanciful  trade  names. 

The  biological  and  mutualistic  relationship  between  the  three  groups  of 
bacteria  and  the  crop  plants  is  very  definite.  The  bacteria  of  group  one 
may  be  looked  upon  as  the  pioneers,  preparing  the  way  for  groups  two 
and  three.  It  is  they  which  prepare  the  raw  and  as  yet  wholly  unavailable 
food  materials  for  the  use  of  growing  crop  plants.  That  is,  they  render 
available  to  the  crop  plant,  either  directly  or  indirectly,  the  as  yet  un- 
available or  locked  foods  of  the  soil.  It  is  they  which  must  contend  with 
the  various  objectionable  organisms  found  in  the  soil,  such  as  the  protozoa 
and  the  molds.  The  bacteria  of  group  two  are  more  favored  by  virtue  of 
their  position  upon  and  near  the  rootlets  and  the  root  hairs  of  plants  where 
the  moisture  supply  as  well  as  chemical  food  materials  are  more  constant. 
Unless  bacteria  of  group  one  perform  their  work  properly,  bacteria  of 
group  two  cannot  in  turn  perform  their  work  and  a  crop  failure  is  the 
result.  Bacteria  of  group  two  may  be  looked  upon  as  the  go  betweens  of 
the  crop  plants  and  the  bacteria  of  group  one.  The  position  as  well  as 
the  work  of  the  bacteria  of  group  three  is  unique.  They  receive  unusual 
protection  by  virtue  of  their  position  within  the  root  nodules  and  they 
deal  directly  with  the  plants  with  which  they  are  thus  closely  associated, 
having  no  biological  relationship  to  the  bacteria  of  either  group  one  or  of 
group  two.  They  constitute  the  transfer  agents  between  the  free  nitrogen 
of  the  air  and  the  host  plants  with  which  they  are  associated.  It  must 
however  be  stated  that  many  bacteria  of  group  two,  as  for  example  the 
Azotobacter  group  (includingal  so  other  organisms  of  the  soil,  such  as 
higher  fungi  and  many  of  the  algae)  have  the  power  of  assimilating,  for 
the  use  of  the  crop  plants,  the  free  nitrogen  of  the  air;  however  free 
nitrogen  assimilation  is  the  special  function  of  the  bacteria  of  group  three. 

The  question  as  to  which  of  the  three  groups  of  bacteria  is  the  most  im- 
portant in  crop  growing,  might  be  debated;  however,  all  scientists  are 
agreed  that  the  three  groups  are  of  the  greatest  importance  in  agriculture. 
Good  crops  might  be  matured  in  soils  having  a  paucity  of  bacteria  of  group 
one  provided  the  necessary  chemical  materials  were  supplied  artificially. 
Again,  fair  crops  might  be  matured  in  soils  having  a  paucity  of  bacteria 
belonging  to  group  two,  provided  certain  other,  directly  available,  chemical 
fertilizers  were  supplied;  and  it  is  known  that  members  of  the  bean  family 
will  grow  to  maturity  and  yield  fairly  well  without  the  root  nodule  bacteria, 

provided  the  proper  nitrogen  bearing  food  materials  are  supplied.     It  has 
11 


1 62  PHARMACEUTICAL  BACTERIOLOGY 

however  been  conclusively  determined  that  all  three  groups  of  bacteria 
are  essential  to  the  best  yield  of  crop  plants. 

Bacteria  of  group  one  are  essentially  rotting  bacteria,  causing  the 
disintegration  of  dead  plants  and  animals,  forming  ammonia  and  other  com- 
pounds. They  are  the  decomposers  of  organic  matter.  Bacteria  of  group 
two  are  essentially  elaborators  of  chemical  compounds  which  the  crop 
plants  can  use  directly  as  food,  in  which  work  these  bacteria  make  use  of  the 
materials  formed  by  the  bacteria  of  group  one.  They  are  therefore  build- 
ers up,  rather  than  tearers  down,  or  decomposers;  although  they  also  form 
certain  decomposition  products.  Bacteria  of  group  three  possess  the 
remarkable  power  of  converting  the  free  nitrogen  of  the  air  into  nitroge- 
nous compounds  which  the  crop  plant  can  utilize  as  food  material. 

As  to  form  (morphology)  most  of  the  soil  bacteria  are  rod  shaped, 
some  being  comparatively  small  and  others  comparatively  large,  but  even 
the  largest  do  not  measure  more  than  9  microns  (a  micron  being  the 
34 >000  Part  °f  one  millimeter)  in  length.  The  smaller  forms  do  not  meas- 
ure over  i  to  3  microns  in  length.  Spherical  forms  also  appear  in  the 
soil  and  upon  the  roots  of  plants.  Spirally  twisted  forms  (Spirillae)  are 
fairly  common  in  most  soils.  As  has  already  been  mentioned,  certain 
highly  pathogenic  bacteria  (disease  producing)  may  be  found  in  soils. 
Old,  long  cultivated  soils  are  apt  to  contain  the  tetanus  bacillus  (the  cause 
of  lock  jaw).  Pasture  lands  may  be  infected  with  the  anthrax  bacillus 
(traceable  to  cattle  dead  from  this  disease).  It  is  believed  that  certain 
animals  (dogs,  coyotes,  wolves)  get  the  dread  disease  rabies  or  hydrophobia 
from  infected  soils,  although  the  usual  source  of  infection  (in  humans  as  well 
as  in  animals)  is  the  bite  of  some  animal  already  suffering  from  this  disease. 

Soil  and  Crop  Bacteria  in  Their  Relationship  to  Cultural  Operations. 
It  has  now  been  conclusively  proven  that  every  agricultural  operation 
which  results  in  an  increase  in  soil  productiveness  or  increase  in  crop  yield, 
has  the  effect  of  encouraging  the  development  of  those  bacteria  which 
elaborate  and  set  free  (render  available  to  the  crop  plant)  those  chemical 
compounds  required  for  the  better  growth  of  the  crop  plants.  Turning 
the  soil  and  making  it  fine  admits  air  and  air  (the  free  oxygen  in  it)  is  one 
of  the  essentials  for  the  development  of  soil  bacteria.  The  rays  of  the  sun 
generate  or  create  warmth  and  warmth  is  the  second  essential  to  the  proper 
development  of  soil  bacteria.  The  other  essential  to  bacterial  develop- 
ment is  moisture  and  every  farmer  knows  that  a  certain  amount  of  soil 
moisture  is  absolutely  essential  for  the  growth  of  plants. 

The  activities  of  soil  bacteria  may  therefore  be  discussed  or  stated 
under  three  heads  as  follows: 

i.  Soil  Moisture  and  Bacterial  Development. — There  are  three  states  or 
degrees  of  soil  moisture  generally  recognized.  First,  that  which  consti- 


BACTERIA   IN   THE   INDUSTRIES  163 

tutes  the  excess  water  (from  rains,  overflows,  melting  snows,  surface  see- 
page) which  percolates  downward  between  the  particles  of  the  soil  (pore 
spaces),  "due  to  the  influence  of  gravity.  This  water  which  is  thus  carried 
downward  more  or  less  rapidly  by  the  force  of  gravity,  is  called  hydrostatic' 
water.  Very  naturally  the  amount  of  hydrostatic  water  in  soils  depends 
upon  the  physical  conditions  of  the  soils  themselves,  more  especially  upon 
the  porosity  and  fineness  of  the  subsoils.  The  hydrostatic  water  carries 
with  it  certain  solutes  (chemical  compounds)  and  very  minute  particles 
(organic,  bacteria,  colloids,  mineral,  etc.)  and  many  of  these  are  wholly 
lost  because  carried  beyond  the  reach  of  the  crop  plants  which  might  have 
utilized  them. 

While  hydrostatic  water  is  instrumental  in  the  distribution  of  soil 
bacteria  it  is  of  little  significance  in  the  growth  and  multiplication  of 
bacteria.  The  crop  soils  should  be  in  such  condition  as  to  allow  the 
hydrostatic  water  to  percolate  to  the  deeper  soil  strata  (two  to  eight 
feet)  and  even  in  these,  deeper  strata  the  pore  spaces  of  the  soil  should 
only  be  partially  filled  with  water  and  never  for  long  periods  of  time 
during  the  growing  season,  because  souring  of  soils  and  root  rot  would 
likely  result,  should  temperature  conditions  be  favorable.  Farmers  in 
arid  and  semi  arid  countries  are  fully  aware  of  the  fact  that  excessive 
irrigation  during  the  growing  season  encourages  root  rot,  especially  of 
the  deep  rooted  crop  plants.  In  fact  heavy  irrigation  during  the  growing 
season  is  one  of  the  means  employed  for  exterminating  the  morning  glory 
and  other  deep  rooted  weeds  (" drowning  out"  the  weeds). 

Hydrostatic  soil  water  is  an  agricultural  essential  as  it  constitutes  the 
storage  water  upon  which  the  growing  plant  must  draw  during  intervals 
of  little  or  no  rain  fall.  The  amount  of  annual  moisture  precipitation 
necessary  to  the  successful  growing  and  maturing  of  crop  plants  depends 
upon  the  nature  of  the  crop  plants,  the  temperature  (during  the  growing 
season),  the  physical  character  of  the  soil,  and  upon  the  methods  and 
means  employed  for  conserving  the  soil  moisture  and  also  upon  the  amount 
of  atmospheric  moisture  present.  Thus  wheat,  barley,  corn,  peas,  beans 
and  other  shallow  rooted  short  period  (six  weeks  to  three  months)  crops, 
can  be  made  to  yield  well  with  a  seasonal  precipitation  not  to  exceed  four  or 
five  inches,  provided,  of  course,  that  the  precipitation  comes  in  one  or 
several  volumes  shortly  before  and  during  the  early  growing  season.  An 
annual  rain  fall  of  from  fifteeen  to  thirty  inches  is  ample  for  the  thrifty 
growth  of  all  kinds  of  plants.  It  is  perhaps  evident  that  the  deeper  soils 
will  retain  and  store  more  hydrostatic  water  than  will  shallow  soils. 
There  are  a  great  number  and  variety  of  factors  which  modify  the  distri- 
bution of  hydrostatic  water. 

It  is  generally  known  that  if  one  end  of  any  dry  porous  substance  (lamp 


164  PHARMACEUTICAL  BACTERIOLOGY 

wick,  piece  of  rope  or  string,  short  piece  of  wood,  etc.)  is  placed  in  water, 
the  moisture  in  the  porous  substance  gradually  rises  above  the  level  of  the 
water.  This  is  due  to  what  is  known  as  capillary  action.  Soil  is  a  porous 
substance  and  the  particles  composing  it  (sand  particles,  bacteria,  organic 
matter,  etc.)  contain  capillary  moisture  derived  from  the  hydrostatic 
water  just  mentioned.  While  hydrostatic  water  invariably  moves  down- 
ward under  the  influence  of  gravity,  capillary  moisture  (or  water)  moves 
exactly  in  the  opposite  direction,  namely  upward,  or,  more  correctly  in  all 
directions  of  the  capillary  influence.  This  capillary  water  or  moisture 
forms  a  continuous  film  over  the  particles  composing  the  soil  and  it  is  in 
this  moisture  that  the  principal  bacterial  development  takes  place.  The 
pores  of  the  soil  in  which  the  capillary  moisture  exists  are  saturated  with 
water  vapor.  As  the  capillary  moisture  evaporates  into  the  air  at  the 
surface  of  the  soil,  new  supplies  are  drawn  upon  from  below  (the  hydro- 
static water),  until  that  supply  is  exhausted,  and  very  soon  thereafter  the 
capillary  moisture  also  disappears  through  evaporation  into  the  air  and 
into  the  soil  pore  spaces.  From  this  it  becomes  evident  that  the  cultural 
operations  should  be  directed  toward  the  reduction  in  the  loss  (by  evapora- 
tion) of  the  capillary  moisture,  which  in  turn  means  the  conservation  of 
the  hydrostatic  water  supply  of  the  soil.  It  only  need  be  stated,  for  the 
purpose  of  explaining  moisture  conservation,  that  the  loss  of  capillary 
moisture  is  completely  checked  as  soon  as  the  air  spaces  about  the  capillary 
or  porous  substance  is  saturated  with  moisture  and  the  escape  of  moisture 
from  such  spaces  is  prevented.  Thus  a  wick  dipped  into  water,  in  a  tightly 
corked  bottle,  will  hold  its  capillary  moisture  indefinitely.  It  must 
however  be  borne  in  mind  that  a  complete  check  in  the  upward  movement 
of  capillary  soil  moisture  would  mean  a  serious  interference  with  the 
bacterial  development  as  these  organisms  require  continually  renewed 
water  supplies  in  order  to  take  therefrom  the  soluble  substances  which  are 
necessary  as  food  materials.  The  cultural  operations  should  therefore  be 
such  as  to  give  rise  to  an  optimum  (rather  than  a  maximum  or  minimum) 
upward  movement  of  capillary  water. 

Soils  which  have  lost  both  hydrostatic  and  capillary  water,  still 
contain  some  moisture  derived  from  the  air  and  this  moisture  is  known  as 
hygroscopic  moisture.  This  varies  in  amount  and  is  contained  in  the 
organic  particles  of  the  soil.  Hygroscopic  moisture  is  generally  not 
sufficient  to  permit  the  development  and  multiplication  of  soil  bacteria, 
but  it  is  sufficient  to  keep  bacteria  alive  as  may  readily  be  proven  by 
making  plate  cultures  of  long  kept  air  dry  soils.  In  some  localities,  the 
hygroscopic  moisture  may  indeed  be  sufficient  to  permit  some  bacterial 
development  in  the  surface  soils,  but  this  moisture  alone  is  inadequate 
to  permit  the  continued  growth  of  crop  plants  or  of  other  larger  vegetation. 


BACTERIA    IN   THE   INDUSTRIES  165 

As  is  known  certain  plants  survive  in  air  dry  soils  because  they  them- 
selves carry  a  surplus  water  supply  upon  which  they  draw  during  the  rainless 
season.  To  this  group  of  plants  belong  the  cacti,  the  palms,  the  cycads 
and  others.  The  plants  with  succulent  or  fleshy  roots,  as  the  radish,- 
beet  and  turnip,  store  water  for  use  during  seasonal  dry  spells.  In  these 
cases  the  bacteria  living  upon  the  root  surfaces,  or  in  the  root  tissues, 
continue  to  multiply. 

The  essentials  of  field  cultural  operations,  as  far  as  bacterial  develop- 
ment and  soil  moisture  are  concerned,  may  be  stated  as  follows.  No 
farm  implement  should  ever  be  allowed  to  operate  in  the  soil  layers  con- 
taining hydrostatic  water.  All  farm  implements  may  work  freely  in 
soils  with  capillary  moisture,  especially  during  the  early  growing  season. 
During  the  actively  growing  season  the  cultivator  should  keep  the  soil 
layers  containing  the  hygroscopic  moisture,  fine  to  very  fine  and  should 
penetrate  through  the  air  dry  layers  into  the  soil  containing  capillary 
moisture,  to  a  depth  of  several  inches.  Keeping  the  top  layer  of  the  soil 
fine  reduces  the  loss  of  moisture  by  evaporation. 

Numerous  scientific  tests  have  been  made  (Russell.  Rothamsted 
Exp.  Station,  England,  and  others)  which  prove  that  soils  which  are  par- 
tially sterilized  by  exposing  them  to  a  temperature  of  60°  C.  improves  the 
productiveness,  due  to  the  fact  that  the  harmful  protozoa  (harmful 
because  they  feed  upon  the  soil  bacteria)  present  are  largely  killed  at 
that  temperature  whereas  the  more  resistent  soil  bacteria  survive  and 
continue  their  beneficent  work  unhindered  by  the  protozoa.  Similar 
results  follow  when  soils  are  exposed  to  certain  antiseptic  vapors,  as  of 
toluene,  benzine,  benzol,  etc.,  which  also  destroy  the  objectionable  pro- 
tozoa without  killing  the  soil  bacteria.  These  undoubtedly  beneficial 
methods  of  partial  sterilization  of  soils  are  not  practicable  on  a  large 
agricultural  scale.  The  farmer  must  therefore  employ  those  methods 
of  cultural  operation  which  will  encourage  to  a  maximum,  the  destruction 
as  well  as  inhibition  of  the  agriculturally  objectionable  protozoa  (including 
also  other  objectionable  organisms),  and  at  the  same  time  encourage  to 
the  optimum  degree  the  growth  and  multiplication  of  the  desirable  soil 
bacteria  (including  also  some  other  organisms).  This,  as  has  already 
been  suggested,  is  accomplished  through  tillage,  drainage,  cultivation, 
etc.  Soils  which  are  excessively  wet,  though  otherwise  satisfactory,  will 
encourage  the  water  loving  protozoa;  and  moist  soils  very  rich  in  humus 
are  apt  to  encourage  the  development  of  the  soD  souring  molds.  Proper 
drainage  and  tillage  prevents  these  troubles. 

2.  Soil  Aeration  and  Bacterial  Development. — The  beneficent  soil  bac- 
teria are  essentially  aerobes  (requiring  free  or  uncombined  atmospheric 
oxygen)  as  already  stated,  and  in  order  that  they  may  develop  to  a 


1 66  PHARMACEUTICAL  BACTERIOLOGY 

maximum  degree,  the  soil  must  constantly  be  supplied  with  air;  in  other 
words,  the  soil  must  be  aerated.  This  is  done  by  stirring  and  turning 
the  soil.  The  stirring  and  turning  operations  must  however  be  of  such 
nature  as  not  to  bring  about  any  undue  waste  or  loss  of  soil  moisture.  As 
soon  as  the  seeds  of  the  crop  plants  have  germinated  and  the  young 
plants  are  growing  actively,  any  deep  turning  of  the  soil  must  cease  as 
this  interferes  with  bacterial  activity  and  furthermore  causes  undue  loss 
of  moisture  by  evaporation.  The  ideal  crop  cultivator  should  keep  the 
soil  (to  a  depth  of  from  three  to  six  inches)  moderately  fine,  well  stirred 
and  should  carry  new  air  into  the  soil.  If  properly  done,  the  only  limit 
to  the  number  of  times  a  crop  plant  may  be  cultivated,  is  of  purely  mechan- 
ical nature.  On  an  average  one  cultivation  every  two  weeks  during  the 
entire  growing  season,  will  perhaps  induce  an  optimum  bacterial  develop- 
ment, and  the  most  desirable  reduction  in  the  rate  of  the  upward  circula- 
tion of  the  capillary  moisture. 

3.  Soil  Temperature  and  Bacterial  Activity. — Not  by  any  means  do  all 
soil  and  plant  bacteria  develop  to  an  optimum  degree  at  the  same  temper- 
ature. Some  of  the  manure  bacteria  develop  actively  at  a  temperature 
of  70°  C.  Some  of  the  bacteria  of  the  ocean  water  and  of  the  polar  regions 
develop  best  at  a  temperature  of  from  10°  C.  to  5°  C.,  and  even  at  lower 
temperatures.  The  great  majority  of  soil  bacteria  in  the  temperate 
regions  develop  most  actively  at  a  temperature  ranging  from  12°  C. 
to  25°  C.  It  is  interesting  to  know  that  while  the  soil  owes  its  warmth 
to  the  heat  rays  of  the  sun,  the  actinic  rays  of  sunlight  inhibit  bacteria) 
development,  especially  in  the  surface  soils,  and  this  reduction  in  bacterial 
growth  in  this  soil  stratum  is  further  increased  by  the  absence  of  capillary 
moisture.  The  maximum  bacterial  development  in  soil  takes  place  at 
a  depth  ranging  from  three  to  six  inches.  Below  that  depth  there  is  a 
gradual  decrease  in  microorganisms,  few  being  found  at  depths  beyond 
four  to  eight  feet. 

In  countries  having  severe  winters  (November  to  April),  the  farmer 
hastens  the  warming  up  of  the  soil  air  in  the  spring,  preparatory  to  crop 
planting,  by  turning  the  soil  with  a  plough.  This  operation  also  causes  a 
better  distribution  of  the  soil  bacteria  and  admits  air.  In  semi-tropical 
and  tropical  countries  the  preparatory  turning  of  the  soil  for  sun  warming 
purposes,  is  not  so  important,  but  proper  soil  aeration  may  nevertheless 
not  be  neglected,  for  reasons  already  stated. 

Bacterial  activity  results  in  some  warmth  generation  and  deep  ma- 
nuring (hot  beds)  is  practised  to  force  young  plants.  The  heat  in  silo 
fermentation  may  rise  to  70°  C.  The  curing  of  hay  and  of  other  vegetable 
substances  results  in  a  rise  in  temperature.  The  bacterial  activities  in 
the  soil  do  not  produce  any  definitely  measureable  rise  in  temperature. 


BACTERIA   IN   THE   INDUSTRIES  167 

In  soils,  the  maximum  bacterial  activity  dependent  upon  temperature, 
is  due  to  the  warmth  created  by  the  sunlight.  At  the  optimum  soil 
temperature,  such  plant  foods  as  ammonia,  nitrates,  phosphates  and  sul- 
phates, are  rapidly  formed.  It  is  perhaps  self-evident  that  soils  in  warm 
countries  furnish  more  available  plant  foods  than  the  soils  in  cold  or 
temperate  countries.  The  production  of  more  plant  food  due  to  increased 
bacterial  activity  results  in  the  more  vigorous  growth  of  higher  plants  and 
this  in  turn  means  the  more  rapid  depletion  of  soil  moisture.  In  warm 
countries  having  rich  soils,  but  with  comparatively  low  annual  rainfall, 
the  conservation  of  soil  moisture  is  of  the  greatest  importance. 

To  sum  up  very  briefly,  soil  bacteria  are  of  the  greatest  importance  in 
agriculture.  The  farm  cultural  operations  are  for  the  purpose  of 
encouraging  the  growth  and  development  of  the  beneficent  soil  and  plant 
bacteria  to  a  maximum  degree.  The  top  soil  must  be  kept  fine  for  the 
purpose  of  checking  the  evaporation  of  the  capillary  moisture.  The 
soil  layers  holding  the  capillary  moisture  must  not  be  too  fine  nor  yet 
too  coarse  and  must  be  frequently  aerated  by  means  of  the  cultivator. 
Wet  soil  must  be  properly  drained  so  as  not  to  allow  the  development  of 
soil  souring  and  otherwise  objectionable  algae,  molds,  protozoa  and  bac- 
teria. If  the  soil  is  too  dry,  there  is  a  paucity  of  desirable  soil  bacteria 
and  the  other  two  groups  of  plant  bacteria  already  mentioned.  If  the 
soil  is  inadequately  aerated,  bacterial  development  is  greatly  reduced. 

Soil  Fertility  and  Bacterial  Activity. — Soil  fertility  is  directly  depend- 
ent upon  the  available  plant  food  which  is  present  and  the  fertility  is  main- 
tained in  two  ways.  By  the  setting  free  (by  chemical  methods)  of  the 
as  yet  unavailable  plant  foods  which  exist  in  the  soil,  and  through  the 
addition  to  the  soil  of  plant  foods  in  the  form  of  fertilizers.  Of  the  latter, 
only  certain  chemical  fertilizers  are  directly  available  to  the  crop  plants, 
others  must  first  be  rendered  available  through  bacterial  activity.  It  is 
true,  all  manures  contain  some  available  plant  food  but  this  also  has  been 
the  result  of  bacterial  activity.  If  follows  as  a  natural  consequence  that 
the  need  for  adding  fertilizers  to  soils  is  reduced  in  the  direct  proportion 
to  the  increase  in  the  development  of  the  normal  and  beneficent  soil  and 
plant  bacteria.  How  long  the  fertility  or  productiveness  of  a  field  may  be 
maintained  by  proper  tillage,  suitable  crop  rotation,  intelligent  summer 
fallowing,  green  manuring,  including  the  rational  use  of  microbial  crop 
inoculation,  has  not  yet  been  determined.  It  is  known  that  soil  has 
yielded  good  crops  for  over  one  hundred  years,  without  the  addition  of 
chemical  fertilizers  or  of  manure.  There  can  be  no  objection  raised  to 
the  intelligent  use  of  fertilizers,  excepting  the  cost  and  the  labor  of  apply- 
ing them.  The  time  honored  practice  of  spreading  a  top  dressing  of 
rich  soil,  from  a  fertile  field,  upon  virgin  soil  (virgin  to  the  crop  under 


1 68  PHARMACEUTICAL  BACTERIOLOGY 

consideration)  and  upon  non-fertile  or  poor  soil,  is  no  longer  considered 
scientifically  correct.  The  transfer  of  such  soils  frequently  caused  trouble 
and  the  harm  done  through  the  simultaneous  transfer  of  objectionable 
soil  fungi,  bacterial  diseases  and  noxious  weeds,  far  exceeded  any  gain 
which  was  derived  from  the  desirable  soil  bacteria  which  were  also  present. 
Bacterial  soil  inoculation  should  be  made  by  means  of  pure  cultures  or 
mixtures  of  pure  cultures,  properly  activated  by  means  of  special  culture 
methods  and  culture  media.  Bacterial  soil  inoculation  is  at  present  merely 
in  its  initial  stages  but  the  results  thus  far  obtained  are  indeed  promising, 
The  results  of  recent  experiments  suggest  the  possibility  that  each  species 
or  kind  of  plant  has  associated  with  it,  via  the  root  system,  certain  bacteria 
which  are  beneficial  and  even  essential  to  its  growth  and  development. 
The  farmer  of  the  near  future  will  no  doubt  inoculate  all  seed,  about  to 
be  placed  in  the  soil,  with  pure  cultures  of  the  predominating  beneficent 
bacteria  belonging  to  the  crop  plant  in  question. 

Soil  Colloids  and  Soil  Fertility. — Within  recent  years  the  chemists 
and  physicists  have  made  certain  very  interesting  observations  regarding 
the  behavior  of  matter  in  very  finely  divided  state  suspended  or  dissolved 
in  liquids  or  in  semi-liquids.  It  has  been  known  for  a  long  time  that  the 
socalled  soluble  salts  (chemical  combinations  of  basic  elements  with  acids) 
when  in  molecular  solution  will  pass  through  dialyzable  membranes  (as 
parchment,  films  of  collodion,  sausage  casings,  dried  and  fresh  skins  of 
animals,  etc.),  whereas  certain  other  substances  apparently  also  in  solution 
(usually  organic  in  nature),  will  not  pass  through  such  membranes.  All 
substances  apparently  in  solution  but  which  will  not  pass  through  dialyz- 
able membranes  are  called  colloids.  Recent  investigations  have  shown 
that  all  substances,  organic  as  well  as  inorganic,  may  be  changed  or  con- 
verted into  colloids.  In  other  words,  colloidality  is  a  universal  property 
of  matter.  Thus,  we  have  colloidal  iron,  colloidal  gold,  copper,  silver, 
mercury,  etc.,  etc.  The  Hindoos  have  for  many  centuries,  prepared  and 
used  colloidal  iron  in  the  treatment  of  certain  diseases,  as  anemia,  and 
other  disorders  in  which  iron  compounds  appear  to  be  deficient.  There 
is  already  a  vast  literature  dealing  with  the  colloids  of  the  soils  in  their 
relationship  to  the  activities  of  soil* bacteria  and  to  soil  fertility.  To 
review,  or  even  to  cite  the  most  important  contribution  to  these  subjects 
would  be  wholly  impracticable  in  this  introduction.  It  is  indeed  regret- 
table that  those  who  treat  of  and  discuss  the  practical  phases  of  cultural 
operations  and  the  use  of  soil  fertilizers,  do  not  explain,  or  at  least  briefly 
indicate,  the  colloidal  principles  involved. 

We  may  therefore,  define  colloids  as  very  minute  particles  composed 
of  groups  of  molecules,  dispersed  in  or  held  in  suspension  in  various  media, 
hcus  as  water  and  other  liquids,  gases,  and  even  in  solids.  It  is  generally 


BACTERIA   IN   THE   INDUSTRIES  169 

believed  that  the  soil  humus  and  soil  colloids  are  one  and  the  same,  but 
this  is  not  in  accord  with  the  facts,  as  may  be  gathered  from  the  above 
introduction.  Humus  is  in  part  colloidal  and  all  humus  has  colloidal 
properties.  The  same  may  be  said  of  the  mineral  constituents  of  the 
soil  no  matter  what  their  composition.  The  agriculturally  active  soil 
colloids  are  represented  by  a  long  list  of  organic  and  inorganic  compounds 
(in  very  minute,  mostly  microscopic  and  ultra-microscopic  molecular 
aggregates)  combined  with  more  or  less  water,  the  water  being  the  chief 
dispersing  medium  for  the  colloidal  particles.  These  soil  colloids  may  be 
roughly  grouped  as  follows: 

1.  Silicic  acid  and  the  soluble  silicates. 

2.  Aluminum  hydroxid  and  its  compounds  with  silicic  acid,  represented 
by  the  clays. 

3.  Iron  hydroxid  and  its  compounds. 

4.  Humus.     Represented  by  humus  acids,  organic  matter  generally, 
including  the  soil  bacteria  and  other  microorganisms  of  the  soil. 

A  fairly  good  idea  as  to  the  nature  of  soil  colloids  may  be  obtained  in 
the  following  manner.  Place  a  teaspoonful  of  rich  soil  in  a  tumblerful 
of  water  and  stir  well,  let  stand  for  a  few  minutes  and  pour  the  superna- 
tant murky  liquid  into  a  second  tumbler  and  let  this  stand  for  thirty  min- 
utes and  again  pour  the  supernatant  liquid  into  a  third  tumbler  and  let 
stand  for  a  few  hours,  decant  very  carefully;  let  stand  for  24  hours  and 
again  decant.  You  will  now  have  five  grades  of  soil  particles.  In  tumblers 
i  and  2  you  will  find  the  coarse  gravel  and  coarser  sand  particles,  with  a 
certain  amount  of  very  fine  matter.  Tumblers  3  and  4  will  contain  the  finer 
sand  particles  and  most  of  the  precipitable  colloids,  while  tumbler  five  will 
retain  the  very  finest  sand  particles  and  the  majority  of  colloids  in 
solution  and  in  suspension.  The  contents  of  tumbler  5  will  appear  quite 
murky  and  will  not  clear  itself  entirely  even  if  allowed  to  stand  undis- 
turbed for  days  and  weeks.  If  this  liquid  should  be  entirely  clarified  by 
filtering  repeatedly  through  a  fine  filter  paper,  it  would  nevertheless 
contain  much  colloidal  matter,  organic  as  well  as  inorganic,  as  might  be 
demonstrated  by  dialysis,  chemical  precipitation,  etc.  The  enormous  river 
deltas  (of  the  Nile  and  the  Mississippi)  are  colloidal  precipitates  the  result 
of  contact  of  the  salt  water  with  the  fresh  river  waters  carrying  the  colloids 
brought  down  from  the  rain  washed  mountain  sides  and  the  valley  lands. 

It  has  been  stated  that  the  fertility  of  the  soils  is  directly  proportional 
to  the  percentage  of  colloids  present.  This  is  certainly  not  true.  An 
excess  of  colloids  is  as  harmful  as  is  a  deficiency  of  colloids.  It  is  therefore 
correct  to  state  that  the  fertility  of  the  soils  is  proportional  to  the  increase 
of  colloids  up  to  the  optimum,  not  the  maximum.  It  has  been  known  for 
centuries  that  the  winter  freezing  of  very  rich  soils  results  in  increased 


170  PHARMACEUTICAL  BACTERIOLOGY 

yield,  due  to  the  coagulation  of  some  of  the  colloids.  The  time  honored 
practice  of  burning  over  soils  excessively  rich  in  organic  colloids  (the  rice 
lands  of  India  and  other  countries)  results  in  increase  in  yield  through  a 
reduction  in  the  organic  colloids.  As  is  well  known  the  rich  soils  of 
California  (adobe  soils)  are  much  improved  by  adding  lime  which  has  the 
effect  of  coagulating  colloids.  The  addition  of  sand  to  heavy  rich  soils  is 
beneficial  because  it  dilutes  the  colloids  and  renders  some  of  them  harmless 
through  adsorption  upon  the  sand  particles.  Adding  phoshates  to  soils 
rich  in  colloids  is  apt  to  result  in  souring  the  soils  because  of  the  increased 
solution  of  the  colloid  (by  the  process  of  hydration) .  Following  the  phos- 
phates with  lime  will  prevent  the  objectionable  changes  through  the  coagu- 
lation of  the  colloids,  as  already  indicated. 

As  the  colloids  increase  beyond  the  optimum  in  wet  soils,  mold  and 
rotting  bacteria  gain  the  ascendency  and  soil  souring  follows,  and  this 
souring  still  further  increases  the  rate  and  degree  of  objectionable  col- 
loidal hydration.  It  is  indeed  true  that  the  water  content  of  the  soils  is 
regulated  through  those  colloids  (organic  as  well  as  inorganic)  which  are 
hydratable,  that  is  which  have  a  very  marked  affinity  for  water.  As  is 
well  known,  gravelly  and  sandy  soils  do  not  hold  the  rain  moisture,  nor  do 
they  readily  draw  the  moisture  from  the  depths  below,  unless  perchance 
the  sand  is  exceedingly  fine. 

One  of  the  important  agricultural  qualities  or  properties  of  colloids 
is  their  power  of  absorption;  that  is,  the  power  of  accumulating  and 
holding  upon  their  surfaces,  the  various  substances  with  which  they  are 
more  or  less  closely  associated  and  which  are  required  by  the  soil  bacteria, 
such  as  water  and  chemicals  inclusive  of  the  smallest  colloidal  particles. 
Unfortunately,  they  adsorp  noxious  gases,  soil  toxins,  bacterial  toxins, 
harmful  chemicals,  and  other  agriculturally  objectionable  substances, 
with  equal  facility,  should  any  be  present.  It  is  the  work  of  the  farmer  to 
so  regulate  and  adjust  the  cultural  operations  as  to  render  impossible,  or 
to  reduce  to  a  minimum,  the  solution  and  adsorption  of  harmful  substances. 

The  growing  crop  plant  cannot  utilize  the  larger  colloidal  particles 
direct.  The  rotting  bacteria,  assisted  by  a  variety  of  enzymes,  transform 
the  colloidal  masses  and  larger  particles  into  smaller  particles,  which  in 
turn  are  seized  upon  by  the  bacteria  of  groups  (2)  and  (3),  converting  them 
into  colloidal  particles  small  enough  to  be  used  as  food  material  by  the 
growing  plant.  The  efforts  of  the  modern  agriculturist  have  to  do,  either 
directly  or  indirectly,  with  the  formation  and  transformation  of  soil 
colloids,  rendering  these  colloids  available  for  the  growing  crop  plant. 
The  change  from  mature  plant  represented  by  straw,  hay,  oats,  corn  etc., 
to  manure,  and  from  manure  to  available  plant  food,  and  from  available 
plant  food  back  into  mature  crop  plant,  is  one  continuous  kalaidoscopic 


BACTERIA  IN   THE   INDUSTRIES 


171 


colloidal  transformation  series,  in  which  water,  enzymes  and  bacteria  play 
the  leading  role. 

Bacterial  Soil  Inoculation — Bacterial  Fertilizers. — By  means  of  thor- 
ough soil  cultivation  and  the  systematic  use  of  fertilizers  we  simply  encour-  - 


PIG.  50. — Longitudinal  section  through  red  clover  rootlet,  showing  tubercle  forma- 
tion due  to  the  root  nodule  microbe,  Rhizobium  mutabile.  The  tubercle  is  only  partially 
developed,  a,  root  hairs.  These  do  not  develop  on  the  nodule,  b,  the  normal  root 
parenchyma,  c,  vascular  tissue,  d,  infected  area,  also  showing  the  infecting  strands 
(Infectionsfaden).  The  cells  are  filled  with  bacteria,  e,  apical  areas,  the  growing  areas 
of  the  tubercle. 


172 


PHARMACEUTICAL  BACTERIOLOGY 


age  the  development  of  the  particular  microbes  that  will  set  free  or  render 
available  the  food  substances  required  by  the  crop  plants  under  cultiva- 
tion. Agricultural  bacteriology  is  beginning  to  make  practical  use  of 
certain  plant  food  forming  microbes.  Of  these  the  free  nitrogen-binding 
microbes  are  most  promising  from  the  standpoint  of  practical  commercial 
utility,  and  have  received  much  attention  in  recent  years.  The  more 
important  species  are:  Rhizobium  mutabile,  Bacillus  ellenbachiensis 
Caron,  Azotobacter  chroococcum;  Bacillus  subtilis,  Bacillus  calif orniensis, 
and  a  few  others.  Of  these,  Rhizobium  mutabtle,  the  root-nodule  bacterium 
of  the  Leguminosae,  has  received  most  attention. 


PIG.  51. — Root  nodules  of  sweet  clover,  somewhat  magnified.  A,  rootlets  with 
nodules,  a,  single  nodules,  b,  clusters  of  nodules.  These  are  sometimes  very  large, 
consisting  of  hundreds  of  nodules,  loosely  united.  B,  diagram  of  single  nodule,  a,  un- 
infected  area,  b,  infected  area. 

The  first  to  suggest  a  plan  for  practically  utilizing  the  root  nodule  bac- 
teria (Rhizobia)  and  to  secure  letters  patent  for  the  process  in  Germany 
and  the  United  States,  were  Nobbe  and  Hiltner,  of  Tharand,  Germany. 
Patent  No.  570,876  was  granted  Nobbe  and  Hiltner  in  the  United  States, 
November  3,  1896.  This  patented  fertilizer  for  leguminous  plants  con- 
sisted of  pure  cultures  of  the  several  varieties  (or  perhaps  species)  of  R. 
mutabile,  each  species  of  plant,  as  bean,  pea,  clover,  alfalfa,  etc.,  having  the 
cultures  derived  from  the  root  nodules  peculiar  to  it. 

This  commercial  preparation  was  given  the  name  "  nitragin,"  and  its 
efficiency  was  quite  carefully  and  extensively  tested  and  commented  upon 
by  European  and  American  investigators.  The  consensus  of  opinion 
seems  to  be  that  it  was  of  doubtful  practical  utility  for  agricultural  pur- 


BACTERIA   IN   THE   INDUSTRIES  173 

poses.  Some  authorities  maintained  that  it  was  of  unquestionable  value  in 
virgin  soil.  In  rich  and  otherwise  favorable  soil  conditions  it  is  of  only 
slight  value.  It  is  maintained  that  nitragin  aids  very  materially_in 
developing  and  ripening  the  fruit.  As  becomes  evident  from  careful  con- 


PIG.  52.  —  Motile  forms  of  Rhizobium  mutabile  as  they  appear  in  fresh  cultures.     They 
are  very  small,  %  to  %/t  in  length. 

sideration,  the  value  of  this  microbic  fertilizer  depends  upon  whether  or 
not  it  will  cause  an  increased  development  in  the  number  and  size  of  root 
tubercles  over  and  above  those  which  would  develop  without  the  presence 
of  this  artificial  aid.  If  the  soil  is  already  well  supplied  with  rhizobia 


FIG.  53. — Non-motile  matured  forms"of\R.  mutabile  as  they  appear  in  mature  sweet 
clover  root  nodules.  Most  of  them^show.the  forked  ends.  This  may  be  considered  the 
normal  form  of  this  organism. 

or  root  tubercle  bacteria,  as  soil  would  naturally  be  if  the  leguminous  plants 
under  consideration  had  been  grown  in  it  for  one  or  more  seasons,  nitragin 
would  in  all  probability  be  of  little  or  no  value.  In  any  case,  the  anti- 
cipated results  have  not  been  fully  realized,  and  nitragin  is  withdrawn  from 
the  market,  and  is  no  longer  manufactured. 


174  PHARMACEUTICAL  BACTERIOLOGY 

A  second  and  later  improvement  in  the  method  of  inoculating  seeds  with 
root  tubercle  bacteria  (Rhizobia)  is  given  by  Hartleb  in  the  specifications 
forming  part  of  letters  patent  No.  674,765,  granted  May  21,  1901,  at  Wash- 
ington, D.  C.  Although  not  so  stated  in  the  specifications,  it  is  evident 
that  the  Hartleb  process  is  a  method  for  applying  pure  rhizobia  cultures  to 
seed  of  leguminous  plants.  Whether  the  method  offers  any  advantages 
over  the  method  of  Nobbe  and  Hiltner  is  questionable.  In  any  case  it 
would  prove  practically  advantageous  only  under  the  conditions  referred 
to  under  the  discussion  of  nitragin.  Although  the  method  has  been  freely 
discussed  and  experimented  upon  in  Germany,  the  fertilizer  is  no  longer 


FIG.  54. — R.  mutabile  as  it  appears  in  mature  nodules  of  red  and  white  clover 
root  nodules.  This  may  be  considered  the  extreme  form  variation  due  to  hyper- 
nutrition. 

on  the  market.  There  is  on  the  market  a  third  patented  germ  or  microbe 
soil  fertilizer  of  German  origin,  known  as  "alinit."  It  consists  essentially 
of  a  pure  culture  of  the  soil  bacillus  known  as  Bacillus  ellenbachiensis 
alpha  or  Bacillus  ellenbachiensis  Caron.  The  germ  was  first  brought  to 
the  attention  of  the  agriculturists  by  Caron,  a  land  owner  of  Germany, 
who  first  isolated  it  and  called  attention  to  the  fact  that  it  had  the  power 
of  chemically  binding  the  free  nitrogen  of  the  air.  The  microbe  is  said 
to  be  closely  allied  to  B.  megatherium  and  B.  subtilis.  According  to  some 
authorities  it  is  especially  concerned  in  assimilating  free  nitrogen  for 
gramineous  plants.  If  it  is  true  it  may  prove  of  great  value  to  grain 
growers. 

The  commercial  alinit  is  a  dry  pulverulent  substance  of  a  yellowish- 
gray  color,  with  about  10  per  cent,  moisture  and  2.5  per  cent,  nitrogen.  It 
is  evidently  prepared  by  mixing  spore-bearing  pure  cultures  of  the  bacillus 


BACTERIA   IN    THE    INDUSTRIES 


175 


FIG.  55. — R.  mutabile  from  the  root  nodules  of  Trifolium  heterodon,  showing  the  ex- 
treme form  variation  due  to  hyper-growth.  The  forms  shown  in  Figs.  52,  53,  54,  55 
and  56  are  simply  in  volution  forms  of  the  same  species  due  to  differences  in  environment 
and  host  relationship.  The  chromatin  bodies  found  in  the  hyper-nourished  forms  (Fig. 
54)  are  probably  reserve  products. 


FIG.  56.  FIG.  57. 

FIG.  56. — Involution  lormc  of  R.  mutabile  as  they  occur  in  artificial  culture  (beef 
broth) .  R.  mutabile  can  be  cultured  quite  readily  upon  a  great  variety  of  culture  media, 
showing  marked  adaptability  to  variations  of  food  supply  and  in  environment. 

FIG.  57. — Azotobacter  agilis  deeply  stained.  This  organism  is  actively  motile  as 
indicated  by  the  pressure  of  numerous  cilia.  The  closely  related  species  A.  chroococcum 
is  less  actively  motile.  Both  possess  the  power  of  free  nitrogen  assimilation  to  a  high 
degree,  especially  when  cultured  in  a  nitrogen-free  medium.  The  organisms  are  large 
(3  to  6/u  in  diameter)  in  the  comparative  sense.  Clostridum  pastorianum  is  also  an  active 
free  nitrogen  assimilator,  but  differs  trom  the  Azotobacters  in  that  if  forms  spores,  a  prop- 
erty which  may  render  it  highly  valuable  in  economic  agriculture  as  cultures  in  the 
sporulating  stage  can  be  kept  for  a  long  time  while  the  cultures  of  non-sporulating  bac- 
teria soon  die  off  or  lose  their  potency. 


176  PHARMACEUTICAL  BACTERIOLOGY 

of  Caron,  with  a  base  of  starch  and  albumen.  It  is  used  to  inoculate  soil 
either  by  spreading  it  broadcast  or  by  sowing  or  otherwise  planting  it  with 
the  seed.  It  is  not  a  nodule  or  root  tubercle-forming  organism,  and  does 
not  enter  into  intimate  symbiotic  or  biologic  relationship  with  plants.  Its 
work  is  simply  that  of  binding  free  nitrogen,  forming  nitrogenous  compounds 
which  enrich  the  soil,  thus  increasing  the  yield  of  any  crop  benefited 
by  such  compounds. 

It  is  known  that  there  are  soil  bacteria  which  are  more  especially  active 
with  certain  plants  or  groups  of  related  plants,  and  this  peculiarity  has 
suggested  the  possibility  of  isolating  them,  artifically  increasing  their 
potency  and  using  them  commercially  for  fertilizing  purposes.  It  is  also 
true  that  not  all. soil  bacteria  are  beneficient.  Under  certain  conditions, 
pathogenic  and  otherwise,  harmful  microbes  are  present  in  great  numbers 
and  become  very  destructive  to  crop  plants,  causing  diseases  of  roots  and 
other  plant  organs.  Bacillus  calif orniensis,  isolated  from  sugar  beets  and 
from  sugar  beet  soil,  appears  to  promote  the  growth  of  sugar  beets,  par- 
ticularly the  seedlings.  The  microbic  leguminous  fertilizer  of  the  Depart- 
ment of  Agriculture,  Washington,  D.  C.,  is  a  slight  modification  of  the 
Hiltner  method.  The  microbic  cultures  are  grown  in  the  absence  of 
nitrogen  or  nitrogenous  compound  making  them  nitrogen  hungry,  thus 
increasing  their  potency  to  produce  nodules  when  brought  in  association 
with  germinating  leguminous  plants.  The  process  is  patented  in  the 
United  States,  and  free  samples  have  been  liberally  distributed  among 
farmers  for  test  purposes,  but  the  results  reported  have  been  rather 
variable,  and  as  a  whole  quite  unsatisfactory.  The  indications  are, 
however,  that  future  experiments  will  clear  up  the  present  difficulties, 
and  some  of  these  so-called  vest-pocket  microbic  fertilizers  will  no  doubt 
prove  highly  beneficial. 

2.    THE  MICROBIOLOGY  OF  WATER  SUPPLIES 

The  following  is  intended  as  an  introduction  to  the  laboratory  methods 
employed  for  the  examination  of  water  supplies.  The  usual  laboratory 
methods  are  chemical  and  bacteriological.  The  bacteriological  methods 
usually  pertain  to  plate  and  tube  cultures  and  have  been  standardized 
and  are  quite  generally  employed  in  state  and  municipal  laboratories. 
Those  interested  are  advised  to  obtain  copies  of  the  methods  as  prepared 
and  used  by  the  Bureau  of  Animal  Industry.  The  official  methods,  that 
is  the  methods  generally  employed  in  the  municipal  and  state  laboratories 
for  the  examination  of  water  supplies,  are  incomplete  as  to  the  micro- 
biological examination,  and  the  organoleptic  tests.  Bacteria  are  not  by 
any  means  the  only  objectionable  organisms  which  occur  in  water,  as  will 
be  explained.  The  microscopical  examination  of  water  has  received  some 


BACTERIA  IN   THE   INDUSTRIES  177 

attention  within  recent  years.  Perhaps  the  best  reference  work  on  this 
phase  of  water  analysis  is  that  by  Whipple  (Microscopy  of  Drinking  Water. 
John  Wiley  and  Sons,  1919)  to  which  the  student  is  referred.  An  excellent 
work  on  water  bacteria  is  that  by  Prescott  and  Winslow  (Elements  of 
Water  Bacteriology.  John  Wiley  and  Sons,  1913).  Most  of  the  authori- 
tative works  on  general  hygiene  contain  a  brief  mention  of  the  micro- 
scopical as  well  as  bacteriological  examination  of  water  supplies. 

Even  the  most  casual  observation  will  make  it  clear  that  the  study 
of  water  supplies  forms  the  very  groundwork  of  sanitary  science.  Not 
only  is  it  important  to  study  the  water  intended  for  drinking  purposes, 
but  also  the  water  supplies  used  for  irrigating  purposes,  the  sewage  waters, 
rivers,  lakes,  ponds,  ditches,  water  of  swamp  lands,  etc. 

A.  Water  Analysis. — Water  and  food  are  the  absolute  essentials  to 
life,  and  of  these  two,  water  is  the  more  important.  The  water  supply 
must  be  ample  and  must  be  of  good  quality.  Water  for  drinking,  cooking 
washing  and  bathing  purposes  must  be  free  from  harmful  or  otherwise 
objectionable  ingredients.  The  water  supplies  intended  for  drinking 
purposes  must  be  free  from  sewage  contaminations  and  must  not  contain 
any  poisons  (chemical,  vegetable,  or  animal)  which  might  prove  harmful. 
The  importance  of  abundant  pure  drinking  water  cannot  be  too  strongly 
emphasized.  An  abundant  supply  must  be  at  hand  or  immediately 
available  at  all  times.  All  possible  desirable  sources  of  drinking  water 
must  be  carefully  investigated  and  examined,  and  the  supplies  well 
guarded  and  none  allowed  to  be  used  for  other  than  drinking  purposes, 
until  that  one  all-important  need  is  liberally  supplied  and  future  require- 
ments assured. 

Streams,  large  and  small,  lakes,  ponds,  wells  and  springs,  are  the  main 
sources  of  drinking  water.  As  a  rule  deep  wells  and  deep  source  springs 
furnish  the  best  drinking  water,  and  shallow  pools  and  small  streams  the 
poorest,  considered  from  the  standpoint  of  possible  harmful  contamination. 
Lakes  and  larger  streams  assure  an  ample  supply  and  the  quality  may  be 
fair  or  even  very  good.  Mountain  streams  fed  by  melting  glaciers  and 
snow,  furnish  good  water. 

Four  methods  are  employed  in  the  examination  of  water,  the  organo- 
leptic,  the  microscopical,  the  bacteriological,  and  the  chemical.  Each 
method  has  its  special  merits  and  all  four  are  essential. 

As  to  the  organoleptic  tests,  water  may  be  wholly  colorless  or  clear;  it 
may  be  turbid  due  to  fine  sand  or  silt  and  clay  or  dirt;  it  may  be  yellowish 
turbid  due  to  fine  clay;  it  may  be  a  yellowish  lemon  tinge  due  to  diatoms; 
or  greenish  due  to  nostoc,  oscillaria  and  other  algae;  it  may  be  brown  or 
flocculent  brown  due  to  iron  fungi  (Leptothrix  and  algae) ;  or  it  may  be 

wine  colored  due  to  peat;  etc.     It  may  be  and  should  be  odorless.     Rain 
12 


178  PHARMACEUTICAL  BACTERIOLOGY 

water  (from  roofs  of  houses)  may  have  a  smoky  (creosote)  odor  or  flavor 
(drained  from  shingled  roofs  of  houses) .  Certain  algae  (Nostoc,  Oscillaria 
and  others)  may  give  rise  to  very  disagreeable  odors.  Chemicals  (perman- 
ganate of  potassium,  hypochlorites,  salt,  lime)  may  impart  a  peculiar 
taste  and  flavor.  The  containers  (buckets,  barrels,  coolers,  wood,  leather, 
canvas,  etc.)  may  transmit  special  flavors.  The  odor  may  be  garlicky 
or  otherwise  disagreeable  due  to  heavy  sewage  contamination.  Pure 
water  is  without  taste,  though  it  does  produce  a  pleasing  gustatary  sen- 
sation if  cool  and  well  aerated.  The  water  in  wells  near  large  bodies  of 
salt  water  (ocean,  bay,  inland  seas)  may  have  a  decidedly  brackish  taste. 
Alkali  seepage  may  impart  a  bitter  or  otherwise  disagreeable  taste. 
The  water  of  small  mountain  streams  is  usually  highly  contaminated  with 
decaying  vegetable  matter  and  frequently  also  contains  decaying  animal 
matter. 

In  the  comparative  sense,  the  highest  contamination  of  bodies  of  water 
prevails  at  or  very  near  the  shore,  no  matter  whether  the  water  is  at  rest 
or  flowing.  The  least  contamination  is  found  at  points  farthest  removed 
from  the  waters  edge,  the  bottom  and  the  surface.  Gravitation  soon  car- 
ries bacteria  to  the  bottom,  while  some  algae  tend  to  accumulate  near  or 
at  the  surface.  In  the  case  of  flowing  water,  the  gradual  retardation  of 
the  rate  of  flow,  toward  the  shore,  causes  an  accumulation  of  deposits  along 
the  outermost  edge  of  the  stream.  The  intake  end  of  the  waterpipe  of 
the  pumping  station  should  be  as  near  to  the  center  of  the  water  supply 
as  possible. 

The  following  is  a  tabulation  of  the  more  important  contaminations 
found  in  water  supplies. 

I.  Derived  from  soils  indicating  surface  waters  and  surface  seepage. 

1.  Vegetable. 

a.  Protococcus  group. 

b.  Desmids. 

c.  Diatoms. 

d.  Nostoc. 

e.  Oscillaria. 

f.  Yeasts. 

g.  Molds. 

h.  Spores  of  cryptogams, 
i.  Pollen  grains, 
j.  Decayed  vegetable  tissues. 

2.  Animal. 

a.  Amebae. 

b.  Paramecia,  Spongilla,  etc. 


BACTERIA   IN   THE   INDUSTRIES  179 

c.  Ova  of  Nematodes. 

d.  Hair  of  animals. 

e.  Insect  fragments. 

f.  Animal  excreta. 

II.  Sewage  Indicators. 

1.  Kitchen  refuse.     Vegetable. 

a.  Starches.     Cereal,  potato,  bean,  etc. 

b.  Spice  elements. 

c.  Vegetable  tissues  derived  from  fruits,  roots,  tubers,  bulbs, 
etc.,  used  as  food. 

2.  Kitchen  refuse.     Animal. 

a.  Blood  corpuscles. 

b.  Oil  and  fat  globules. 

c.  Muscle  elements. 

d.  Fibrous  tissue  elements. 

e.  Ova  of  intestinal  parasites. 

3.  Bacteria.   : 

a.  Coccus  forms,  abundant. 

b.  Diplobacilli,  usually  abundant. 

c.  Streptococcus  fecaliSj  usually  present.     A  positive  sewage 
indicator. 

d.  A  positive  presumptive  colon  bacillus  test. 

4.  Other  organisms. 

a.  Yeasts  and  mold  may  be  present. 

b.  Spores  of  molds  and  ova  of  intestinal  parasites  may  be 
present. 

c.  Motile  protozoa. 

III.  Mineral  and  inorganic. 

1.  Oil  or  resinoid  matter  derived  from  vegetable  decay  (pines). 

2.  Dirt  and  clay  particles. 

3.  Sand  particles.     Coarse  and  fine.     Colorless  and  colored. 

4.  Mineral  particles  and  particles  of  mineral  compounds.     Color- 
less and  with  color.     Iron  compounds. 

5.  Diatomaceous  earth.     Kaolin.     Etc. 

IV.  Amorphous  organic  particles.     These  are  present  in  direct  proportion 
to  organic  contamination. 

The  analyst  should  note  the  following  regarding  possibly  available 
sources  of  water  supply. 

1.  The  character  of  the  underlying  geologic  formation. 

2.  The  surrounding  vegetation  and  the  animals  that  dwell  in  the 
vicinity. 


l8o  PHARMACEUTICAL  BACTERIOLOGY 

3.  The  human  habitations  near  the  supply. 

4.  The  source  of  the  water. 

5.  The   available   amount   of   the   supply.     Cubic    contents,    or 
volume  of  flow  per  hour. 

6.  All  possible  sources  of  contamination  and  the  nature  of  the 
contamination. 

A  microscopical  examination  should  be  made  of  the  shore  and  bottom 
mud  and  ooze  at  the  points  of  most  likely  contamination  and  a  record  made 
of  the  microscopic  flora  and  fauna  of  the  shore.  If  the  body  of  water 
is  small  and  is  highly  contaminated  by  cattle,  horses,  or  hogs,  it  must  be 
abandoned  for  drinking  purposes,  or  the  water  first  purified  by  the  use  of 
some  coagulant,  by  filtration  and  by  boiling.  If  the  body  of  water  is 
large  and  the  shore  is  contaminated  by  animals  the  water  must  be  taken 
from  a  middle  point  and  either  boDed  or  treated  with  hypochlorites,  or 
other  chemicals.  Samples  should  be  taken  by  means  of  suitable  sampling 
bottles  and  some  of  the  water  thus  collected  should  be  centrifuged  and 
the  sediment  examined  microscopically  and  the  findings  recorded. 

After  the  completion  of  the  topographical  survey,  the  organoleptic 
tests,  and  the  microscopical  examination,  the  analyst  is  then  in  position 
to  make  a  definite  recommendation  as  to  what  is  to  be  done  regarding 
the  water  in  question.  His  recommendations  will  be  along  the  following 
lines. 

1.  Unfit  for  drinking  purposes  and  impossible  or  impracticable  to 
render  it  potable. 

2.  Suitable  for  drinking  purposes  in  the  raw  state.     Safe.     No  treat- 
ment required. 

3.  Suitable  for  drinking  purposes,  after  filtering  through  sand  filter. 

4.  Suitable   for  drinking  purposes,  after  coagulating  and  filtering. 
Sand-alum  filtration.     Sedimentation  and  filtration. 

5.  Suitable  for  drinking  purposes  after  adding  calcium  hypochlorite. 
Hypochlorous  acid  sterilization  of  suspicious  water  supplies. 

Additional  methods  for  purifying  water  supplies  are. 

1.  The  use  of  Pasteur-Chamberland  pressure  filters.     These  are  im- 
practicable for  large  volumes  of  water.     May  be  applicable  in  homes 
public  buildings  and  hospitals. 

2.  Use  of  gravity  porous  clay  filters  and  coolers.     Often  quite  satis 
factory  where  the  quantities  of  water  used  are  comparatively  small,  as  ii 
private  homes,  stores,  schools,  etc. 

3.  Distilling  the  water.     Rather  impracticable  for  large  volum< 
Used  on  board  ships. 


BACTERIA   IN   THE   INDUSTRIES  l8l 

4.  Boiling  the  water  for  30  minutes.    Very  satisfactory  for  rendering 
contaminated  waters  entirely  safe  for  drinking  purposes. 

5.  Ultra-Violet  Rays. — The  high  voltage  ultra-violet  ray  mercury  lamp 
has  been  used  quite  successfully  by  the  French  army  in  the  sterilization~6f 
drinking  water.     The  lamps  are  of  quartz  which  permit  the  more  effective 
passage  of  the  ultra-violet  rays,  and  are  placed  directly  into  the  water  and 
are  said  to  have  a  radius  of  action  of  one  foot  and  sterilization  is  said  to  be 
complete  in  one  minute  without  resulting  in  any  physical  changes  in  the 
water.    The  rays  are  not  effective  in  heavily  polluted  waters  nor  in  water 
which  is  not  clear.    Heavily  polluted  waters  may  first  be  clarified  and 
partially  purified  by  precipitation  (the  alumn  method)  and  filtration 
(through  sand,  charcoal,  cotton,  etc.). 

6.  Centrifugal  Purification. — The  centrifuge  has  recently  come  int 
extensive  use  for  the  purpose  of  clarifying  and  purifying  liquid  substances 
of  various  kinds,  as  gelatinous  solutions,  plant  juices,  syrups,  oils,  paints, 
varnishes,  beers,  wines,  etc.     High  speed  turbine  driven  machines  are  now 
upon  the  market  making  from  24,000  to  40,000  revolutions  per  minute 
(the  Sharpies  Centrifuge).     The  Sharpies  machine  is  of  the  continuous 
feed  type  which  may  be  operated  by  hand  (25,000  revolutions)  or  by 
either  steam  or  compressed  air  (40,000  revolutions).     This  machine  will 
render  water  absolutely  clear  no  matter  how  heavily  polluted,  and  it  is 
said  to  remove  most  of  the  bacteria.     In  case  of  water  which  is  suspected  of 
containing  disease  germs  (typhoid,  cholera,  etc.)  the  centrifuging  must  be 
followed  by  chemical  or  ultra-violet  ray  sterilization. 

The  analyst  will  find  details  for  the  application  of  the  sand-alum 
filtration  and  the  use  of  calcium  hypochlorite  in  Field  Hygiene  by  Ford 
and  in  Sanitation  for  Medical  Officers  by  Vedder  and  in  other  works  on 
sanitation. 

The  analyst  should  be  thoroughly  familiar  with  the  subject  of  water 
purification  and  should  assist  the  sanitary  officers  in  their  work.  Steriliza- 
tion of  drinking  water  by  means  of  chemicals  has  been  very  carefully 
worked  out  by  the  armies  in  Europe.  The  following  chemicals  have  been 
used. 

1.  Chlorinated  lime  (chloride  of  lime)  with  35  per  cent,  available 
chlorine   gas.    Action   depends   upon   the   hypochlorous   acid   formed. 
Amount  used  ranges  from  1-1,000,000  to  1-25,000.     A  chlorinated  lime 
containing  75  per  cent,  available  gas  is  on  the  market  and  is  preferable  to 
the  weaker  lime. 

2.  Sodium  hypochlorite  is  more  efficient  and   also  more  expensive. 
Use  about  the  same  amount  as  of  chlorinated  lime. 

3.  Sodium  bisulphate  in  tablets,  30  grains  to  the  quart  of  water,  shake, 


1 82  PHARMACEUTICAL  BACTERIOLOGY 

and  let  stand  for  20  minutes.     Excellent  for  small  quantities,  as  for  cavalry 
men,  and  for  men  on  the  march. 

4.  Potassium  permanganate,  i  grain  to  the  quart,  or  enough  to  pro- 
duce   a   pinkish    coloration.      Particularly  efficacious    against  Bacillus 
choleras. 

5.  Calcium  permanganate  is  used  in  Germany  to  purify  water  in  the 
canteens  (i  grain  to  the  quart).     The  precipitate  which  forms  must  be 
filtered  through  plug  of  cotton  or  a  filter  paper  cap. 

6.  Iodine  liberated  from  the  mixing  of  iodide  and  iodate,  in  the  canteen, 
has  been  used  by  the  French  army.     The  salts  are  put  up  in  red,  white,  and 
blue  tablets,  ready  for  immediate  use. 

7.  Halazone   (p-sulphon  dichloramino  benzoic  acid).     1-300,000  is 
very  efficient  against  typhoid  and  cholera  contaminations.     This  is  cheap 
and  effective.     It  comes  in  tablets  ready  for  use. 

The    following    titrating   method   for   determining   the   amount   of 
chlorine   required   to   sterilize  water  supplies  is  used  in  the  army,  as 
reported  by  Vedder  (Sanitation  for  Medical  Officers,  Lea  and  Febiger, 
.1917). 

1.  Into  a  rinsed  ordnance  cup  (i  pint  or  about  500  cc.  capacity)  break 
one  tube  of  calcium  hypochlorite  and  mix  thoroughly  with  a  few  drops  of 
water.     Fill  the  cup  with  water  to  within  one  inch  of  the  top  (500  cc.) 
and  mix  well  by  pouring  back  and  forth  by  means  of  a  second  cup.     This 
solution  contains  0.3  gram  of  available  chlorin. 

2.  Rinse  four  ordnance  cups  with  the  water  to  be  sterilized  and  fill 
all  four  cups  with  water  to  be  tested.     To  first  cup  add  0.2  cc.  of  the 
test  solution  in  cup  mentioned  in  (i).     To  second  cup  add  0.4  cc.,  to 
third  o.  6  cc.  and  to  the  fourth  cup  0.8  cc.     Mix  well  by  pouring  back  and 
forth.    Let  stand  for  thirty  minutes. 

3.  Into  a  clean  cup  crumble  a  tablet  of  potassium  iodide  (or  use  a  few 
crystals  of  the  iodide  salt),  add  a  little  starch  solution  (made  by  boiling 
a  little  corn  starch).     Pour  into  this  cup  the  water  to  which  was  added 
0.8  cc.  of  the  chlorite.     If  blue  color  appears,  it  is  an  indication  that  not  all 
of  the  chlorine  has  been  used  up  in  that  mixture. 

4.  The  cup  which  contains  the  smallest  amount  of  the  chlorite  which 
will  give  a  blue  color,  contains  the  percentage  of  chlorine  required  to 
sterilize  the  water  to  be  used. 

5.  Example. — Let  us  suppose  that  the  cup  to  which  was  added  the  0.4 
cc.  hypochlorite  solution  represents  the  smallest  amount  just  producing  a 
blue  coloration,  hence  enough  to  sterilize  one  pint.     To  sterilize  36  gal- 
lons (288  pints,  the  amount  in  one  Lyster  water  bag)  would  require  115 
cc.   of  the  test  solution  in  cup  (i).     The  pint  of  hypochlorite  prepared 
would  therefore  suffice  to  sterilize  a  little  over  four  bags  of  water.     In 


BACTERIA   IN   THE    INDUSTRIES  183 

practice  it  is  customary  to  use  double  the  amount  indicated  by  the 
reaction. 

The  caution  to  be  observed  in  the  use  of  chemical  water  sterilizers  is 
to  mix  them  thoroughly  into  the  water  and  allow  them  sufficient  time  to 
act,  at  least  20  minutes.  The  amounts  used  should  be  adjusted  to  the 
degree  of  contamination  which  is  ascertained  by  titration. 

It  must  be  borne  in  mind  that  filtering  material,  no  matter  what  kind, 
soon  becomes  clogged  with  accumulated  sediment  and  rapidly  developing 
algae  and  fungi.  The  filtering  material  must  therefore  be  frequently 
renewed,  in  some  cases  every  day  and  in  no  case  should  the  interval  be 
more  than  five  days.  The  analyst  should  make  daily  examinations  of  the 
accumulated  scum  as  this  will  convey  valuable  information  as  to  the 
impurities  in  the  water  and  the  efficiency  of  the  filter.  The  filter,  -if  not 
properly  attended  to,  may  itself  become  a  source  of  contamination. 
Crenothrix  forms  are  very  apt  to  develop  in  sand  filters,  especially  if  there 
is  a  deficiency  in  oxygen  supply. 

Water  supplies  (ponds,  small  lakes)  with  abundant  filamentous  algae 
may  be  treated  with  copper  sulphate  (1-1,000,000)  before  filtration. 
This  method  met  with  much  favor  a  few  years  ago,  but  has  been  quite 
abandoned  and  forgotten  recently.  The  copper  sulphate  can  be  placed  in 
a  gunny  sack,  fastened  to  a  boat,  and  distributed  by  rowing  the  boat  about 
until  all  of  the  chemical  is  dissolved,  i  part  in  4,000,000  has  given  good  re- 
sults, as  far  as  freeing  the  water  of  filamentous  algae  and  also  protozoa,  is 
concerned.  After  filtration  no  copper  can  be  found  in  the  water.  The 
filtering  material  must,  of  course,  be  renewed  occasionally. 

Sedgwick  and  Rafter  have  devised  a  method  of  making  a  microscopical 
examination  of  water.  Desirable  quantities  of  water  are  run  through  a 
graduated  cylinder,  having  a  sand  plug  at  the  lower  open  end.  In  place  of 
this  cylinder  an  ordinary  glass  or  tin  funnel  may  be  used.  Place  a  per- 
forated stopper  (cork  or  rubber)  at  lower  end,  carrying  bent  outflow  tube. 
On  top  of  opening  in  stopper  place  a  bit  of  cotton  or  cheesecloth,  followed 
by  ignited  sand  quartz  (sands,  Nos.  60,  120,  140,  as  may  be  desired). 
On  top  of  the  sand  place  another  bit  of  cotton  or  cheesecloth.  Pour 
through  the  desired  volume  of  water  (250  cc.,  500  cc.,  1000  cc.,  etc.), 
and  then  mix  the  sand  (with  the  cotton  and  cloth)  in  5  or  10  cc.  of 
distilled  or  pure  water  and  examine  microscopically  making  the  desired 
counts. 

The  following  blank  report  sheet  of  a  bacteriological  examination 
will  serve  to  illustrate  the  nature  of  such  analysis  in  a  state  or 
municipal  laboratory: 


184  PHARMACEUTICAL  BACTERIOLOGY 

Report  of  the  Bacteriological  Examination  of  Water 


Laboratory  No Date  Reported 

Town  or  locality 

Source  of  water : 

Location  of  sampling  point 

Collected  by Date 

Reported  to 

Number  Bacteria  per  cc..  Gelatin  20°  C Agar  37.5°  C 

B.  Coli confirmed  in cc.     B.  Coli  Index,  approximate  number  per  cc. 

Turbidity Alkalinity  as  CaCO3 Chlorine Hardness. . . 

(Results  in  parts  per  million) 

Condition  of  sample: 
Remarks: 

Approved: 


Chemist  and  Bacteriologist  Director,  Bureau  of  Sanitary  Engineering 

EXPLANATION  OF  RESULTS 

The  laboratory  results  can  be  properly  interpreted  only  in  the  light  of  a  compre- 
hensive field  examination  of  the  source  of  the  water  by  an  expert.  Laboratory  tests 
can  not  show  whether  colon  bacilli  in  the  water  were  derived  from  the  excreta  of  animals 
or  the  much  more  dangerous  sewage  carrying  the  feces  of  human  beings.  Neither  can 
the  laboratory  determine  whether  a  slight  pollution  at  one  time  would  mean  a  heavy 
pollution  at  another,  owing  to  fluctuations  in  the  volume  of  sewage  or  to  seasonal  varia- 
tions in  the  amount  of  water,  disturbance  of  sediment  by  waves,  etc.  Careless  sampling 
and  growth  in  transit  are  large  factors. 

Certain  conclusions  can,  however,  be  drawn  from  the  laboratory  reports  alone.  If 
colon  bacilli  are  not  demonstrable  in  10  cc.  of  the  water,  it  may  be  regarded  as  safe 
for  drinking  purposes  at  the  time  of  the  taking  of  the  sample,  as  far  as  sewage  pollution 
and  consequent  danger  from  typhoid  fever  and  other  water-borne  diseases  is  concerned. 
If  colon  bacilli  are  demonstrable  in  .10  cc.,  but  not  in  i  cc.  the  water  may  ;be  looked 
upon  as  under  suspicion,  and  a  field  examination  is  necessary  to  determine  whether  or 
not  it  is  safe.  If  colon  bacilli  are  found  in  i  cc.  of  the  water,  but  not  in  o.i  cc.  the 
water  should  be  regarded  as  probably  unsafe,  and  a  purification  process  should  be  in- 
stalled if  the  sources  of  contamination,  animal  or  human,  can  not  be  removed.  If 
colon  bacilli  are  found  in  o.i  cc.  of  the  water,  or  smaller  amounts  the  water  should  be 
considered  as  polluted  to  such  a  point  as  to  be  unsafe  for  drinking  purposes. 

The  B.  Coli  Index  is  the  reciprocal  of  the  smallest  portion  of  water  in  which  the  B. 
Coli  group  is  confirmed.  The  number  of  B.  Coli  in  a  reasonably  safe  water  should  be 
less  than  o.i  cc. 

3.     BACTERIA  IN  MILK  AND  IN  THE  DAIRYING  INDUSTRY 
A.  General  Discussion 

Bacteria  play  an  important  part  in  modern  dairying,  and  they  are 
destined  to  play  even  a  more  significant  part  in  the  near  future.  Certain 
microbes  are  active  in  the  ripening  of  cream,  butter  and  cheese.  Formerly 
it  was  customary  to  let  nature  attend  to  the  inoculation  of  the  cheese, 
resulting  in  a  rather  variable  product.  Now  the  up-to-date  dairy-man 


BACTERIA   IN    THE    INDUSTRIES  185 

inoculates  the  cheese  with  pure  cultures  of  the  kind  of  microbe  producing 
the  desired  flavor  as  Roquefort,  Bre,  Limburger,  etc.  In  time  it  will  no 
doubt  be  possible  to  produce  hitherto  unheard-of  cheese  flavors  by  means 
of  new  species,  varieties,  and  strains  of  cheese  microbes.  Cream- and 
butter-flavor  bacteria  are  also  used.  The  souring  of  milk  is  due  to  the 
omnipresent  but  illy  defined  Bacillus  acidi  lactici  and  other  bacteria. 
Stringy  or  ropy  milk  is  due  to  bacterial  infection.  Under  conditions 
favorable  to  the  development  of  the  organisms,  the  ropiness  appears 
within  from  twelve  to  twenty-four  hours  after  milking,  and  becomes  so 
pronounced  that  the  milk  can  be  drawn  out  in  long  threads  or  strings. 


PIG.  58. — -Lactic  acid  bacillus.  There  is  a  large  group  of  bacteria,  similar  in  appear- 
ance to  the  lactic  acid  bacillus,  which  have  the  power  of  forming  lactic  acid  in  milk. 
Some  of  these  are  used  in  pure  culture  to  make  the  so-called  artificial  buttermilk.  Milk 
bacteriology  is  still  in  its  infancy.  For  so  long  have  we  been  accustomed  to  the  use  of 
contaminated  (filthy)  milk  that  in  a  recent  test  made  with  samples  of  pure  milk  and 
samples  of  which  cow  manure  was  added,  90  per  cent,  of  those  who  were  asked  to  taste 
the  milks  preferred  the  milk  to  which  the  cow  manure  was  added,  declaring  that  it 
was  the  only  sample  which  had  a  "milk  flavor." 

It  is  a  not  uncommon  condition  of  milk  in  Switzerland,  where  is  is  con- 
sidered specially  noxious,  but  in  Holland  it  has  been  produced  by  design 
for  making  Edam  cheese.  Ropiness  of  milk  is  caused  by  a  variety  of 
microorganisms,  among  them  being  Bacillus  actinobacter,,  B.  lactis  vis- 
cosus,  B.  gummosus,  etc.  The  microorganism  used  in  Holland  for  the 
manufacture  of  the  cheese  referred  to  is  known  as  the  Streptococcus  Hoi- 
landicus.  The  Bacillus  cyanogenus  causes  the  milk  to  become  blue  with- 
out coagulating  it  or  rendering  it  acid.  The  Bacillus  butyricus  occurs  in 
milk  which  it  coagulates,  also  producing  butyric  acid.  It  is  this  microbe 
which  develops  the  rancidity  of  butter.  There  are,  however,  many  dif- 
ferent species  of  microbes  which  produce  butyric  acid  fermentation. 


1 86  PHARMACEUTICAL   BACTERIOLOGY 

Freshly  drawn  milk  is  not  germ-free,  even  under  the  most  aseptic  and 
sanitary  conditions  and  surroundings.  As  a  rule  even  the  milk  in  the 
udder  contains  some  germs,  in  spite  of  the  fact  that  milk  possesses  de- 
cidedly bactericidal  properties.  However,  the  milk  from  different  animals 
varies  in  this  regard.  The  bacterial  impurities  of  freshly  drawn  milk  are 
traceable  to  the  skin  of  the  cow,  the  dust  and  filth  about  cow  stables,  the 
vessel  containing  the  milk,  and  above  all  to  the  hands  of  the  milkers. 
The  milker  is  often  the  cause  of  inoculating  the  milk  with  disease  germs, 
as  typhoid, acute  dysentery,  diphtheria,  scarlet  fever,  small-pox,  and  tuber- 
culosis. The  medical  journals  cite  cases  of  typhoid  epidemics  traceable 
to  milkers  who  were  "typhoid  carriers"  without  actually  suffering  from 
the  disease.  Cows  are  very  susceptible  to  tuberculosis,  and  the  milk  from 
tuberculous  animals  has  infected  thousands  upon  thousands  of  children 
and  many  adults. 

Since  milk  is  an  excellent  culture  medium  for  a  great  variety  of  germs, 
it  is  evident  that,  under  favorable  conditions,  it  may  be  a  fruitful  source 
of  infections.  Serious  epidemics  of  typhoid  fever  and  of  diphtheria  have 
been  traceable  to  and  exactly  limited  to  the  milk  route  of  a  certain  dairy- 
man. Tuberculous  infections  of  the  children  in  a  number  of  families  have 
been  traceable  to  the  milk  from  a  single  animal.  As  a  rule  mixed  milk 
(that  is  the  milk  from  many  animals)  is  safer  than  the  milk  from  a  single 
animal,  though  this  is  not  necessarily  always  the  case.  The  milk  from 
animals  that  are  free  from  disease  and  that  are  tested  regularly  once  each 
year)  for  tuberculosis,  and  that  are  kept  under  sanitary  conditions,  is 
absolutely  safe,  provided  the  containers  are  clean  and  the  milkers  and 
others  in  the  dairying  establishment  are  free  from  latent  or  active  com- 
municable disease  and  are  cleanly  in  their  habits.  The  number  of  germs 
in  freshly  drawn  milk  varies  from  1000  to  several  millions  per  cc.,  and  is 
directly  proportional  (within  the  limits  indicated)  to  the  cleanliness  and 
sanitary  conditions  of  the  dairying  establishment.  The  bacterial  content 
of  milk  from  the  same  source  is  of  course  higher  in  warm  and  hot  weather 
than  it  is  in  cold  weather,  other  things  being  equal.  Certain  dairying 
establishments  supply  what  is  known  as  "certified  milk,"  or  milk  which  ij 
certified  by  the  board  of  health  as  coming  from  animals  that  are  regularly 
tested  for  tuberculosis  and  which  are  kept  under  the  sanitary  conditions 
imposed  by  the  milk  commission  or(  by  the  board  of  health,  furthermore, 
such  milk  must  be  bottled  in  sterilized  bottled  which  are  hermetically 
sealed  and  placed  on  ice  at  once  and  kept  on  ice  until  delivered  to 
consumer.  There  is,  however,  a  lack  of  uniformity  in  the  regulations 
governing  the  supply  of  certified  milk  in  different  communities.  The 
following  conditions  should  prevail: 

a.  All  cows  should  be  healthy,  that  is,  free  from  diseases  of  all  kinds. 


BACTERIA    IN    THE    INDUSTRIES  187 

The  animals  should  be  tested  for  tuberculosis  every  six  months.  As  soon 
as  an  animal  gives  a  positive  reaction  for  tuberculosis,  it  should  be  removed 
from  the  herd  and  killed.  Milk  from  sick  animals  should  not  be  used.^ 

b.  The  sanitary  conditions  and  environment  of  pasture,  grazing  lands, 
sheds,  stables,  etc.,  should  be  excellent.     The  entire  water  supply  should 
be  pure,  and  all  water  supplies  should  be  tested  chemically  and  bacterio- 
logically  at  suitable  intervals.     All  food  supply  for  cows  must  be  whole- 
some and  free  from  objectionable  contaminations. 

c.  Those  employed  about  the  establishment  must  be  free  from  latent  or 
active  disease.     They  should  be  tested  for  tuberculosis,  latent  typhoid, 
and  should  be  examined  for  skin  diseases.     They  must  be  cleanly  in  their 
habits.     Before  milking,  the  hands  of  the  milkers  and  the  teats  of  the 
animals  should  be  washed  with  clean  warm  water  and  then  dried  with  a 
clean  towel. 

d.  The  containers  must  be  sterilized  thoroughly  every  day,  inside  and 
outside.     This  can  be  done  by  thoroughly  washing  and  rinsing  in  boiling 
hot  water  and  thoroughly  drying,  before  pouring  milk  into  them. 

e.  Just  as  soon  as  the  milk  is  drawn,  it  should  be  bottled  (sterilized 
bottles),  bottles  capped,  hermetically  sealed  (paraffin),  and  placed  on  ice 
until  ice-cold,  and  delivered  at  once  to  the  consumer.     The  bottles  should 
be  on  ice  in  delivery,  and,  even  though  hermetically  sealed,  should  be  kept 
away  from  dust  and  dirt.     The  bottles  should  be  placed   in   paper 
bags  so  that  the  driver  need  not  touch  them  at  all.     The  housewife 
should  take  the  bottle  from  the  bag  and  place   it   in   the   ice-chest, 
cellar,  or  cooler  until  the  milk  is  wanted  for  use. 

Milk,  on  standing,  should  show  no  dirt  deposit.  This  crude  test  is  a 
fairly  reliable  guide  as  to  the  sanitary  conditions  in  the  dairying  establish- 
ment and  the  rules  of  cleanliness  that  are  observed.  It  has  been  shown 
that  the  quantity  of  bacteria  in  freshly  drawn  milk  is  directly  proportional 
to  the  amount  of  dirt  (sediment)  present.  A  bottle  or  tumbler  full  of  milk 
should  show  no  dirt  sediment  after  standing  for  an  hour  or  longer. 

Good  cows'  milk  should  have  from  3.5  to  4.0  per  cent,  of  butter  fat. 
It  is  marketed  in  three  forms:  Full  milk  having  all  of  the  butter  fat,  half 
milk  or  partially  skimmed  milk,  and  skimmed  milk.  Because  of  the 
variability  of  milk  which  is  partially  skimmed,  it  would  be  wise  to  with- 
draw it  from  the  market.  When  milk  is  sold  without  further  specification, 
full  or  unskimmed  milk  is  understood.  It  is  unlawful  to  sell  skimmed 
milk  as  milk,  or  without  designating  it  as  skimmed  milk. 

In  some  countries,  as  Germany  for  example,  the  rules  and  regulations 
directed  against  dairies,  dairying  and  the  sale  of  milk,  are  very  far-reaching, 
and  are  strictly  enforced  by  the  local  health  authorities.  Specific  rules  are 
laid  down  as  to  what  milk  may  or  may  not  be  marketed,  how  the  cows  are 


1 88  PHARMACEUTICAL  BACTERIOLOGY 

to  be  kept,  what  cattle  diseases  render  the  milk  unfit  for  use,  how  cows  and 
milkers  must  be  prepared  for  the  milking  process,  etc.  The  use  of  pre- 
servatives is  not  permitted,  because  these  substances  reduce  the  digesti- 
bility of  the  milk  and  because  their  use  encourages  lax  and  careless  methods 
in  the  dairying  establishments. 

The  bovine  disease  most  to  be  dreaded  is  tuberculosis.  It  is  very  prev- 
alent among  cattle,  and  the  milk  from  tuberculous  cows  is  a  serious  menace 
to  the  health  of  those  who  use  it,  particularly  to  susceptible  (by  inheritance 
children.  The  most  efficient  means  of  safeguarding  the  public  health 
against  this  source  of  infection  consists  in  removing  the  infected  animals 
from  the  herd,  with  a  view  to  disposing  of  them  by  slaughter  and  burial 
as  soon  as  circumstances  will  permit.  Where  this  wasteful  method  has 
been  employed  the  results  have  been  discouraging,  even  when  the  State 
recompensed  the  owner  in  part  for  the  loss  of  his  stock.  The  government 
meat  inspection  regulations  now  admit  the  use  of  meat  of  slightly  tuber- 
culous animals,  for  it  is  declared  that  under  such  circumstances  the 
thorough  cooking  of  meat  is  an  effective  safeguard  against  danger. 

Testing  cows  for  the  presence  of  latent  or  undeveloped  forms  of  tubercu- 
losis is  simple,  safe,  and  should  be  rigidly  persisted  in.  Turberculin  is  in- 
jected into  the  neck  or  shoulder  region.  If  tuberculosis  exists  there  will  be 
a  rise  in  temperature  (102°  to  104°  F.),  in  the  course  of  from  eight  to  eighteen 
hours.  If  the  disease  is  far  advanced  there  may  be  no  reaction,  in  fact,  the 
reaction  is  then  unnecessary  as  the  indications  are  already  sufficiently 
positive. 

The  tuberculin  used  is  prepared  from  glycerinated  bouillon  in  which 
tubercle  bacilli  have  been  grown  from  six  to  eight  weeks.  The  bouillon 
culture  is  first  boiled  for  two  hours  to  kill  all  the  living  organisms.  It  is 
then  filtered  under  pressure  through  a  germ-proof  earthenware  filter  to  re- 
move the  dead  bodies  of  the  germs,  concentrated  by  evaporation,  a  little 
carbolic  acid  added,  and  it  is  then  bottled  for  distribution.  There  is  no 
evidence  that  its  use  causes  an  increase  in  the  rapidity  of  the  progress  of 
the  disease  in  animals  already  affected  with  tuberculosis,  or  that  it  is 
injurious  to  them  in  any  other  way.  It  does  not  even  temporarily  injure 
the  quality  of  the  milk. 

Preservatives,  as  boric  acid,  salicylic  acid,  benzoic  acid,  sodium  benzo- 
ate  and  formalin,  are  sometimes  added  to  milk  to  prevent  bacterial  develop- 
ment. A  very  small  amount  of  formalin  (1:10,000)  is  sufficient  to  check 
the  souring  of  milk.  The  others  are  added  in  larger  amounts  (1:1000  or 
more).  These  additions  are  not,  as  a  rule,  appreciable  through  the  sense 
of  taste  or  smell  and  do  not  in  any  way  modify  the  appearance  of  the  milk. 
In  some  countries  milk  preservatives  are  permissible,  in  others  they  are 
not,  and  in  still  others  they  are  permitted  provided  there  is  a  declaration 


BACTERIA   IN   THE   INDUSTRIES  189 

to  that  effect  and  the  amount  does  not  exceed  a  definite  percentage,  as 
provided  by  law. 

In  England,  a  limited  amount  of  certain  preservatives  added  to  milk  is 
permissible,  the  argument  being  that  it  is  better  to  supply  preserved  milk 
than  milk  loaded  with  germs.  This  argument  has  its  commendable 
features.  In  very  large,  congested  cities  like  London,  New  York  and 
Chicago,  it  is  impossible  to  supply  the  poor  with  certified  milk  or  milk 
which  can  be  kept  free  from  excessive  germ  development  until  it  is  wanted 
for  consumption. 

Boiling  the  milk  for  twenty  minutes  kills  the  germs,  but  unfortunately 
the  boiling  temperature  produces  certain  changes  which  greatly  reduce 
the  food  value  of  the  milk,  besides  the  germicidal  properties  of  the  milk 
are  destroyed,  so  that  the  bacterial  development  is  afterward  even  more 
active  than  before.  Sterilizing  at  lower  temperature  (50°  to  80°  C.)> 
known  as  pasteurizing,  does  not  interfere  with  the  nutritive  qualities  of 
the  milk,  but  destroys  the  bactericidal  properties,  as  already  mentioned. 
The  process  is,  however,  generally  recommended  by  physicians.  A  simple 
home  method  may  be  carried  out  as  follows  (Roger) : 

Milk  is  most  conveniently  pasteurized  in  the  bottles  in  which  it  is 
delivered.  To  do  this  use  a  small  pail  with  a  perforated  false  bottom.  An 
inverted  pie  tin  with  a  few  holes  punched  in  it  will  answer  this  purpose. 
Punch  a  hole  through  the  cap  of  one  of  the  bottles  and  insert  a  thermometer. 
Set  the  bottles  of  milk  on  the  pie  tin  in  the  pail  and  fill  the  pail  with  water 
nearly  to  the  level  of  the  milk.  Put  the  pail  on  the  stove  or  over  a  gas 
flame  and  heat  it  until  the  thermometer  in  the  milk  shows  not  less  than 
65°  C.  nor  more  than  70°  C.  The  bottles  should  then  be  removed  from  the 
water  and  allowed  to  stand  from  twenty  to  thirty  minutes.  The  tempera- 
ture will  fall  slowly,  but  may  be  held  more  uniformly  by  covering  the 
bottles  with  a  towel.  The  punctured  cap  should  be  replaced  with  a  new 
one,  or  the  opening  sealed  with  wax  or  paraffin,  or  the  bottle  may  be 
covered  with  an  inverted  cup. 

After  the  milk  has  been  held  as  directed  it  should  be  cooled  as  quickly 
and  as  much  as  possible  by  setting  in  water.  To  avoid  danger  of  breaking 
the  bottle  by  a  too  sudden  change  of  temperature,  this  water  should  be 
warm  at  first.  Replace  the  warm  water  slowly  with  cold  water.  After 
cooling,  milk  should  in  all  cases  be  kept  at  the  lowest  available  temperature. 

It  should  be  remembered  that  pasteurization  does  not  destroy  all 
bacteria  in  milk,  and  after  pasteurization  it  should  be  kept  cold  and  used 
as  soon  as  possible. 

Rosenau  sums  up  the  pros  and  cons  of  milk  pasteurization  as 
follows : 

Advantages.— The  advantage  of  pasteurization  is  that  it  is  a  cheap  and 


190  PHARMACEUTICAL  BACTERIOLOGY 

effective  means  of  preventing  the  transmission  of  infectious  diseases  such 
as  tuberculosis,  typhoid  fever,  diphtheria,  scarlet  fever,  etc.,  commonly 
spread  by  milk. 

Disadvantages. — a.  Pasteurization  promotes  carelessness  on  the 
farm  and  dairy,  etc.  (This  may  be  controlled  by  proper  regulations,, 
inspections  and  laboratory  examinations.) 

b.  Pasteurization  renders  milk  less  digestible.     (While  it  is  generally 
conceded  that  boiled  milk  commonly  induces  constipation,  the  majority  of 
the  evidence  plainly  indicates  that  pasteurization  has  little,  if  any,  effect  on 
the  digestibility  of  the  milk.) 

c.  Pasteurized  milk  favors  the  production  of  rickets  and  scurvy. 
(There  is  no  proof  to  this  effect  and  authorities  agree  that  the  danger  is 
slight;  and,  further,  that  it  may  readily  be  obviated.) 

d.  By  destroying  the  non  spore-bearing  bacteria,  pasteurization  some- 
times allows  toxic  organisms  to  grow  and  produce  serious  poisons  in  the 
milk.     (On  the  other  hand,  these  same  poisons  are  more  frequently  pro- 
duced in  milk  that  has  not  been  pasteurized,  and  thus  danger  may  be 
obviated  in  pasteurized  milk  by  cooling  it  quickly,  keeping  it  cold  and 
shortening  the  time  for  distribution.) 

e.  Pasteurization  is  inefficient  as  a  preservative;  the  milk  keeps  only 
twelve  to  twenty-four  hours  longer  than  otherwise.     (This  is  really  no 
disadvantage,  for  the  quicker  bad  milk  sours,  the  better.) 

/.  Pasteurization  injures  the  taste  of  the  milk.  (This  is  not  so,  if 
properly  done.) 

g.  Pasteurization  increases  the  cost  of  the  milk.  (True,  but  it  is  the 
cheapest  safeguard,  and  the  expense  of  pasteurization  is  offset  by  the 
keeping  quality  of  the  milk.) 

Rosenau  has  made  extensive  tests  to  determine  the  thermal  death- 
point  of  those  pathogenic  microbes  most  commonly  found  in  milk.  His 
conclusions  are  summarized  as  follows: 

Milk  heated  to  60°  C.  and  maintained  at  that  temperature  for  two- 
minutes  will  kill  the  typhoid  bacillus.  The  great  majority  of  these 
organisms  are  killed  by  the  time  the  temperature  reaches  59°  C.,  and  few 
survive  to  60°  C. 

The  diphtheria  bacillus  succumbs  at  comparatively  low  temperatures. 
Oftentimes  it  fails  to  grow  after  heating  to  55°  C.  Some  occasionally  sur- 
vive until  the  milk  reaches  60°  C. 

The  cholera  vibrio  is  similar  to  the  diphtheria  bacillus  regarding  its 
thermal  death-point.  It  is  usually  destroyed  when  the  milk  reaches 
55°  C.;  only  once  did  it  survive  to  60°  C.  under  the  conditions  of  the 
experiments. 

The  dysentery  bacillus  is  somewhat  more  resistant  to  heat  than  the 


BACTERIA    IN    THE    INDUSTRIES  IQI 

typhoid  bacillus.  It  sometimes  withstands  heating  at  60°  C.  for  five 
minutes.  All  are  killed  at  60°  C.  for  ten  minutes. 

So  far  as  can  be  judged  from  the  meager  evidence  at  hand,  60°  C.  for 
twenty  minutes  is  more  than  sufficient  to  destroy  the  infective  principle  T)f 
Malta  fever  in  milk.  M .  melitensis  is  not  killed  at  55°  C.  for  a  short  time; 
the  great  majority  die  at  58°  C.,  and  at  60°  C.  all  are  killed. 

Milk  heated  to  60°  C.  and  maintained  at  that  temperature  for  twenty 
minutes  may,  therefore,  be  considered  safe  so  far  as  conveying  infection 
with  the  microorganisms  tested  is  concerned. 

Evaporated,  condensed  and  dry  milk  are  found  upon  the  market  and 
are  extensively  used.  Sugar  is  frequently  added  as  a  preservative.  In 
making  condensed  milk,  it  is  evaporated  in  large  pans  until  it  assumes  a 
creamy  consistency.  Dry  milk  is  prepared  by  spraying  the  milk  on  revolv- 
ing hot  cylinders.  The  thin  film  of  milk  is  evaporated  to  dryness  in  a 
moment,  and  in  that  state  is  scraped  from  the  cylinders.  Dry  milk  is  a 
common  ingredient  of  baby  foods  and  invalid  foods,  and  is  also  very 
extensively  used  in  the  manufacture  of  chocolate  creams.  The  condensed 
and  dry  milks  do  not  keep  long  in  spite  of  the  greatest  care  in  manufacture. 
The  containers  and  milk  must  be  .thoroughly  sterilized  or  pasteurized, 
and  the  cans  must  not  be  opened  until  ready  for  use.  Such  preservatives 
as  salicylic  and  boric  acid  are  sometimes  added  to  condensed  milk. 

It  is  known  that  sweet  cream  yields  a  very  insipid,  flavorless  butter, 
whereas  cream  which  has  " soured"  for  a  few  days  yields  a  pleasant  tasting 
and  pleasingly  flavored  butter,  provided  the  desirable  species  or  variety  of 
bacteria  are  present.  If  the  souring  is  continued  too  long  the  flavor  may 
be  hopelessly  vitiated.  In  the  past  it  was  customary  to  add  a  smaU  amount 
of  old  cream,  having  a  desirable  flavor,  to  a  new  lot  of  cream.  This  mother 
cream  was  designated  the  '' starter."  It  contained  the  desirable  cream- 
ripening  bacteria,  mostly  of  the  lactic  acid  variety.  These  old-time  natural 
starters  are  now  largely  replaced  by  starters,  prepared  in  the  laboratory 
consisting  of  pure  cultures  of  certain  strains  or  varieties  of  cream  flavor, 
producing  germs  of  the  lactic  acid  group.  A  proper  regulation  of  the 
temperature  is  very  important  in  the  ripening  of  cream  (60°  to  75°  F.). 
It  is  also  necessary  to  pasteurize  the  cream  before  adding  the  bacterial 
starter  in  order  to  prevent  the  development  of  microbes  which  might 
interfere  with  the  proper  development  of  the  starter  microbes.  Naturally 
the  use  of  clean,  sterilized  utensils  and  uniformity  of  methods  are  all- 
important,  in  order  that  uniform  results  may  be  obtained. 

Cheese  flavors  are  also  due  to  bacterial  action,  but  not  wholly  so, 
as  many  of  the  higher  fungi,  as  species  of  Penicillium  (Camembert  Penicil- 
lium)  and  of  Oidium  (O.  lactis)  also  play  a  very  important  part  as  flavor 
producers.  The  Roquefort  cheese  owes  its  characteristic  flavor,  in 


IQ2  PHARMACEUTICAL  BACTERIOLOGY 

part  at  least,  to  a  variety  or  form  of  Penicillium  glaucum.  The  qualities 
and  properties  of  some  Swiss  and  soft  Belgian  cheeses  are  largely  due  to 
Oidium  lactis.  The  ripening  of  hard  cheeses  (Cheddar,  Edam,  American, 
some  Swiss  varieties,  and  others)  is  due  exclusively  to  bacterial  action. 
Cream,  butter  and  cheese  are  very  prone  to  the  attacks  of  objectionable 
bacteria  and  moulds  which  cause  very  unpleasant  flavors  and  bitter  taste. 
It  must  also  be  borne  in  mind  that  cream,  cheese  and  butter  may  carry 
disease  germs.  Tubercle  bacilli  have  been  reported  in  these  food  arti- 
cles, but  it  has  not  been  demonstrated  that  they  are  frequently  present. 
Typhoid  infections  have  been  traced  to  the  use  of  cream,  but  no  case  of 
typhoid  fever  has  ever  been  definitely  traced  to  eating  butter  or  cheese. 
Of  course,  these  articles  may  become  infected  after  manufacture  and  thus 
become  a  possible  means  of  spreading  disease. 

B.  The  Bacteriological  and  Microscopical  Examination  of  Milks 

The  examination  and  rating  of  milk  is  largely  a  municipal  affair  and 
every  city  of  any  considerable  size  has  a  Board  of  Health  or  an  officer 
who  is  empowered  to  enforce  the  regulation  governing  the  quality  of  the 
milk  to  be  sold.  Dealers  in  milk  and  dairy  men  are  required  to  produce 
evidence  that  they  are  complying  with  the  health  ordinances  pertaining 
to  the  sale  of  milk  for  human  consumption.  Score  cards  are  provided 
which  the  keepers  of  dairies  must  fill  out.  Evidence  as  to  the  conditions 
under  which  the  cows  are  kept  must  be  furnished.  Samples  are  taken 
from  time  to  time  and  analyzed  in  the  city  laboratory,  which  examination 
is  generally  chemical  and  bacteriological.  The  legal  definitions  of  the 
different  kinds  of  milk  are  given  and  the  analysts  are  required  to  show 
whether  or  not  the  quality  of  the  milk  under  examination  conforms  to  the 
definition.  The  following  statements  will  make  these  points  clear. 

i.  General  Statement  as  to  Quality. — The  lacteal  secretion  obtained 
from  the  domesticated  cow  and  from  other  animals,  as  goat,  mare,  and 
ass,  is  a  highly  important  food  article.  The  chief  food  ingredients  which 
it  contains  are  casein,  fat,  and  sugar  (lactose).  The  major  bulk  is  water. 
Milk  happens  to  be  an  ideal  food  for  many  bacteria  and  raw  milk  freely 
exposed  to  air  undergoes  complete  bacterial  decomposition  in  a  compara- 
tively short  time,  the  rate  of  decomposition  depending  on  temperature, 
oxygen  supply  and  degree  of  contamination.  The  organisms  which 
may  be  considered  as  the  normal  milk  decomposers  are  the  group  of  lactic 
acid  formers,  or  the  milk  sourers.  These  appear  to  be  omnipresent  in 
the  air  and  upon  the  earth's  surface,  entering  air  exposed  milks  and  by 
their  rapid  multiplication  soon  crowd  out  other  associated  air  bacteria. 

The  production  of  pure  milk  is  all-important  and  pure  milk  can  be 


BACTERIA   IN   THE   INDUSTRIES  1 93 

•supplied  provided  sanitary  rules  and  regulations  are  strictly  observed  and 
enforced. 

Temperature  and  climatic  conditions  are  of  great  importance  in 
regulating  the  bacterial  contamination  of  milks.  Thus  there  is  in  many 
states  and  localities,  a  summer  standard  and  a  winter  standard.  Again, 
the  varied  and  variable  dairying  conditions  has  resulted  in  the  recognition 
-of  several  grades  of  milk,  as  certified,  guaranteed,  Grade  A,  Grade  B,  etc. 

The  milk  from  diseased  animals  is  universally  recognized  as  objec- 
tionable and  no  one  would  knowingly  use  such  milk.  Milk  also  serves  as  a 
carrier  of  disease,  such  as  tuberculosis,  various  forms  of  coccic  and  strepto- 
coccic  infection,  typhoid,  dysentery,  diphtheria,  etc.  These  are  all  matters 
of  general  knowledge  to  sanitarians  and  need  not  be  discussed  more  fully. 

2.  Milk  Impurities.— Under  the  compound  microscope,  ordinary  cow's 
milk  may  show  the  following  elements. 

a.  Butter  fat  globules.     Variable  in  size,  occurring  singly  and  more  or 
less  agglutinated.1 

b.  Casein  granules.     Very  minute  and  formless,  granular. 

c.  Body  cells.     Epithelial  cells,  leucocytes,  red  blood  corpuscles,  pus 
corpuscles. 

d.  Impurities  derived  from  cow,  from  cow  stable,  from  cattle  feed. 

e.  Impurities  derived  from  milkers. 

f.  Impurities  derived  from  containers. 

g.  Impurities  derived  from  air  and  dust. 

h.  Pathologic  impurities  from  the  milk  yielding  animals  and  from  the 
human  associates. 

As  far  as  the  micro-analytical  examination  of  milk  is  concerned,  no 
attempt  is  made  to  identify  the  different  species  of  microorganisms  which 
may  be  present.  The  essential  is  to  determie  whether  or  not  the  milk 
in  question  is  fresh,  pure,  and  wholesome. 

3.  General  Milk  Rating. — Two  distinct  milk  ratings  must  be  noted* 
The  older  rating  based  upon  plate  and  tube  cultures,  still  operative  or 
applied  in  most  laboratories,  and  the  more  recent  rating  based  upon  direct 
microscopical  examination.     The  older  method  (plating  method)  gives 
evidence  of  the  approximate  number  of  living  bacteria  present  only  and  is 
quite  limited  in  scope  and  significance.     The  direct  microscopical  examina- 
tion gives  evidence  as  to  the  following. 

1.  Total  number  of  dead  and  living  bacteria  present. 

2.  Body  cells  of  all  kinds. 

3.  Colostrum  (fat  globules  and  characteristic  ameboid  body  cells). 

4.  Dirt  and  similar  impurities  present. 

5.  Butter  fat  present. 

1  Boiling  and  pasteurizing  causes  the  fat  globules  to  agglutinate. 
13 


1 94  PHARMACEUTICAL  BACTERIOLOGY 

The  following  extracts  from  the  California  State  Pure  Milk  Act  ex- 
plains the  essentials  regarding  pure  milk. 

1.  Tuberculin  Test. — It  shall  be  unlawful  for  any  person,  firm  or  cor- 
poration, except  in  bulk  to  the  wholesale  trade,  to  sell  or  exchange  or 
offer  or  expose  for  sale  or  exchange  for  human  consumption  any  milk  from 
cows  that  have  not  passed  the  tuberculin  test,  until  it  has  been  pasteurized 
by  the  holding  process  at  a  temperature  not  less  than  one  hundred  forty 
degrees   Fahrenheit  for  twenty-five  minutes;  provided,   that    milk  for 
drinking  purposes  shall  not  be  heated  above  one  hundred  forty-five 
degrees  Fahrenheit.     It  shall  further  be  unlawful  for  any  person,  firm  or 
corporation  to  sell  or  exchange  or  offer  or  expose  for  sale  or  exchange  any 
milk  products  except  cheese,  into  the  composition  of  which  any  milk 
enters  other  than  that  permitted  in  this  section  of  this  act,  to  be  sold  at 
retail.     For  the  purpose  of  this  act  milk  shall  be  construed  to  include 
cream. 

2.  Uninspected  Milk. — It  shall  be  unlawful  for  any  person,  firm  or 
corporation  to  sell  or  exchange,  or  offer  for  sale  or  exchange,  in  any  city, 
country  or  city  and  county,  in  which  a  milk  inspection  service,  approved 
by  the  state  diary  bureau,  has  been  established,  any  milk  otherwise  than 
as  hereinafter  provided  in  this  act,  and  for  the  purpose  of  this  act,  the  term 
"inspecting  department"  shall  be  construed  to  mean  the  health  depart- 
ment of  a  county  or  group  of  counties,  city  or  group  of  cities,  or  city  and 
county,  maintaining  a  milk  inspection  service  approved  by  the   state 
dairy  bureau. 

3.  Impure  Milk. — All  milk,  except  certified  milk,  guaranteed  milk, 
grade  A  milk,  and  grade  B  milk,  is  hereby  declared  to  be  impure  and 
unwholesome  and  must  not  be  sold  for  human  consumption. 

4.  Grades. — For   the   purpose  of  this  act,  milk  shall  be  graded  as 
follows:     certified  milk,  guaranteed  milk,  grade  A  milk,  grade  B  milk 
and  milk  not  suitable  for  human  consumption;  provided,  that  milk 
not  suitable  for  human  consumption  shall  be  plainly  so  marked. 

5.  Guaranteed    Milk. — No   person,  firm  or  corporation  shall  sell  or 
exchange,  or  offer  or  expose  for  sale  or  exchange,  as  or  for  guaranteed  milk, 
any  milk,  raw  or  pasteurized  the  quality  of  which  is  guaranteed  by  the 
dealer,  without  approval  in  writing  of  the  inspecting  department,  which 
milk  must  be  of  a  higher  standard  than  that  required  for  grade  A  raw  milk. 

6.  Grade  "A"  Milk. — No  person,  firm  or  corporation  shall  sell  or 
exchange,  or  offer  or  expose  for  sale  or  exchange,  as  and  for  grade  A  milk 
any  milk  that  does  not  conform  to  the  rules  and  regulations  and  the 
methods  and  standards  for  production  and  distribution  of  grade  A  milk 
adopted  by  the  inspecting  department. 

Grade  A  milk  shall  conform  to  the  following  requirements  as  a  mini- 


BACTERIA   IN    THE    INDUSTRIES  1 95 

mum :  if  raw,  it  shall  consist  of  the  clean  raw  milk  from  healthy  cows  as 
determined  by  physical  examination  and  by  the  tuberculin  test  by  a  quali- 
fied veterinarian  under  the  supervision  of  the  inspecting  department,  and 
from  dairies  that  score  not  less  than  seventy  per  cent,  on  the  score  card  - 
adopted  by  the  United  States  bureau  of  animal  industry,  department  of 
agriculture.  The  tuberculin  test  must  be  repeated  annually  if  no  reacting 
animals  are  found  in  the  herd.  If  reacting  animals  are  found  they  must 
be  removed  from  the  herd,  and  the  tuberculin  test  repeated  in  six  months. 
All  cows  are  to  be  fed,  watered,  housed  and  milked  under  conditions  ap- 
proved by  the  inspecting  department.  All  persons  who  come  in  contact 
with  the  milk  must  exercise  scrupulous  cleanliness  and  must  not  harbor 
the  germs  of  typhoid  fever,  tuberculosis,  diphtheria,  or  other  infectious 
diseases  liable  to  be  conveyed  by  milk.  Absence  of  such  infections  shall 
be  determined  by  cultures  and  physical  examination,  to  the  satisfaction 
of  the  inspecting  department. 

This  milk  is  to  be  delivered  in  sterile  containers  and  is  to  be  kept  at  a 
temperature  established  by  the  inspecting  department  until  it  reaches 
the  ultimate  consumer,  when  it  must  contain  less  than  one  hundred  thou- 
sand bacteria  per  cubic  centimeter.  If  pasteurized  it  shall  come  from  cows 
free  from  disease  as  determined  by  physical  examination  at  least  once  in 
six  months,  by  a  qualified  veterinarian  of  an  inspecting  department.  It 
shall  contain  less  than  two  hundred  thousand  bacteria  per  cubic  centimeter 
before  pasteurization  and  less  than  ten  thousand  bacteria  per  cubic  centi- 
meter at  the  time  of  delivery  to  the  ultimate  consumer.  Dairies  from 
which  this  milk  is  derived  must  score  at  least  sixty  on  the  score  card 
adopted  by  the  United  States  Bureau  of  animal  industry,  department 
of  agriculture. 

7.  Grade  "  B"  Milk. — No  person,  firm  or  corporation  shall  sell  or 
exchange,  or  offer  or  expose  for  sale  or  exchange,  as  and  for  grade  B  milk, 
any  milk  that  does  not  conform  to  the  following  requirements  as  a  mini- 
mum: it  must  be  obtained  from  cows  in  no  way  unfit  for  the  production  of 
milk  for  use  by  man,  as  determined  by  physical  examination  at  least  once 
in  six  months  by  a  qualified  veterinarian  of  an  inspecting  department. 
Before  pasteurization  such  milk  shall  contain  less  than  one  million  bac- 
teria per  cubic  centimeter.  After  pasteurization  it  shall  contain  less  than 
fifty  thousand  bacteria  per  cubic  centimeter. 

Milk  for  pasteurization  must  be  kept  at  a  temperature  established  by 
the  inspecting  department  up  to  the  time  of  delivery  to  the  pasteurization 
plant  and  rapidly  cooled  after  pasteurization  to  a  temperature  of  fifty 
degrees  Fahrenheit,  or  below,  and  so  maintained  to  the  time  of  delivery 
of  the  same.  Pasteurization  shall  be  by  the  holding  method  at  a  tem- 
perature not  less  than  one  hundred  forty  degrees  Fahrenheit;  provided, 


PHARMACEUTICAL  BACTERIOLOGY 


that  milk  for  drinking  purposes  shall  not  be  heated  above  one  hundred 
forty-five  degrees  Fahrenheit. 

Such  pasteurizing  plant  shall  be  equipped  with  a  self -registering  device 
for  record  of  the  time  and  temperature  of  pasteurization.  Such  records 
shall  be  kept  for  two  months  and  be  available  for  inspection  by  any  health 
department,  the  state  veterinarian  or  any  of  his  agents,  or  the  state  dairy 
bureau.  Pasteurized  milk  shall  be  marked  with  the  day  of  the  week  of 
pasteurization  and  must  be  delivered  to  the  consumer  within  forty-eight 
hours  thereafter.  If  milk  is  repasteurized,  it  must  not  be  sold  except 
as  not  suitable  for  human  consumption. 

8.  Milk  Not  Suitable  for  Human  Consumption.- — Milk  not  suitable  for 
human  consumption  may  be  sold  for  industrial  purposes,  provided  it  be 
heated  to  a  higher  temperature  than  necessary  for  pasteurization,  and 
delivered  in  a  distinctive  container,  plainly  marked  with  the  words  "Not 
suitable  for  human  consumption,"  in  letters  not  less  than  one-quarter 
inch  in  length  and  one-twelfth  inch  stroke. 

The  following  limit  counts  are  based  upon  direct  microscopical  ex- 
amination and  is  intended  as  a  guide  to  analysts. 

TOTAL  NUMBER  PER  cc. 


Grades 

Bacteria 

Body  cells 

Organic  particles 

Pat  globules 

Ordinary 

50  ooo  ooo 

qjOO  OOO 

I  OOO  OOO 

Certified  

20,000,000 

4.O.OOO 

<;  0,000 

2,000,000,000 

Guaranteed  
Grade  A... 

25,000,000 
20  000,000 

4O,OOO 
AO.OOO 

50,000 
<o,ooo 

to 

4,500,000,000 

Grade  B  

50,000,000 

4O,OOO 

75,000 

s/ 


Canned  Milk 

The  usual  laboratory  routine  in  the  examination  of  canned  milks  is 
chemical,  ascertaining  total  solids,  -butter  fat,  lactose,  sucrose,  etc.  The 
usual  quality  standards  for  evaporated  milks,  including  the  standard  of  the 
United  States  Bureau  of  Chemistry,  are  based  upon  chemical  composition 
rather  than  upon  means  for  ascertaining  possible  organic  contamination. 
The  Bureau  of  Chemistry  Standard  specifies  that  "  evaporated  milk  should 
be  prepared  by  evaporating  fresh,  pure  milk  obtained  from  healthy  cows. " 
The  following  method  will  make  it  possible  to  determine  the  freshness,, 
purity,  and  wholesomeness  of,  or  the  bacterial  and  other  contamination  ia 
evaporated  milk,  after  such  milk  has  been  canned,  processed,  and  offered 
on  the  market. 

While  the  processing  is  intended  to  and  does  kill  all  organisms  which 
may  be  present  in  the  milk,  it  does  not  destroy  or  decompose  them,  or 


BACTERIA   IN    THE   INDUSTRIES  1 97 

render  them  invisible  or  unrecognizable  under  the  compound  microscope. 
The  microscope  will  therefore  reveal  all  of  the  microorganisms,  and  also 
other  contaminations,  which  were  introduced  or  which  developed  in  the 
milk  prior  to  and  up  to  the  very  moment  of  the  final  processing. 

The  following  findings  and  recommendations  pertain  to  evaporated 
milks,  whole  and  skimmed,  sweetened  and  unsweetened : 

1.  The  organisms  most  commonly  present  are 

a.  Cocci, 

b.  Diplococci, 

c.  Diplobacilli, 

d.  Streptococci, 

e.  Bacilli, 

f.  Tetracocci  (Sarcina), 

naming  them  in  the  order  of  their  relative  abundance.  Of  these  groups, 
the  diplococcus,  diplobacillus,  and  streptococcus  forms  are  the  most  im- 
portant from  the  viewpoint  of  the  food  bacteriologist. 

2.  Many  of  the  coccus  and  diplococcus  forms  are  no  doubt  derived 
from  the  streptococcus  forms.     Because  of  the  fact  that  coccus  forms  may 
be  confused  with  minute  fat  globules  and  nonbacterial  organic  particles 
of  spherical  form,  it  is  suggested  that  the  coccus  count  be  omitted. 

3.  The  diplobacillus  forms  thus  far  observed  are  readily  confused 
with  diplococcus  forms,  because  the  individual  cells  are  but  slightly 
elongated,  the  two   diameters  being  as  i  :  1.3.     It  is  therefore  recom- 
mended that  the  diplococcus  and  diplobacillus  forms  be  included  in  one 
and  the  same  count. 

4.  Bacilli  are,  as  a  rule,  sparingly  and  irregularly  present  in  evaporated 
milks.     The  same  may  be  said  of  the  tetracoccus  forms. 

5.  It  does  not  appear  practicable  to  make  body  cell  counts  of  evapo- 
rated milks,  and  it  is  recommended  that  this  be  omitted  as  a  laboratory 
routine. 

6.  The  amount  of  organic  debris  present  in  evaporated  milk  is  quite 
variable  in  different  cans  of  a  given  brand  of  canned  milk.     It  is,  however, 
recommended  that  milk  be  examined  as  to  the  relative  amount  of  organic 
debris  present,  bearing  in  mind  that  the  careless  or  inexperienced  micro- 
analyst  may  confuse  casein  particles,  agglutinated  butter   fat  globules 
and  lactose  crystals,  with  organic  debris. 

7.  Organoleptic  testing  is  of  considerable  importance  in  milk  examina- 
tion.    Consistency,  color,  odor,  and  taste,  should  be  carefully  noted. 

8.  It  is  also  self-evident  that  the  presumptive  colon  bacillus  test  should 
give  negative  results  with  all  heat-sterilized  milks.1 

1  It  is  a  notable  fact  that  pasteurization  does  not  always  kill  the  colon  bacilli  in  milk. 
Numerous  samples  of  pasteurized  milk  have  been  found  which  subsequently  contained 
practically  pure  cultures  of  the  colon  bacillus. 


198  PHARMACEUTICAL    BACTERIOLOGY 

The  following  recommendations  are  made: 

A.  That  the  quality  and  purity  of  sterile  evaporated  milks  be  based 
upon  the  following  findings  given  in  order  of  their  importance: 

a.  Diplobacilli, 

b.  Diplococci, 

c.  Streptococcus  chains, 

d.  Organic  debris,  other  than  casein  or  butter  fat. 

It  is  recommended  that  the  diplococcus  and  diplobacillus  forms  be 
included  in  one  and  the  same  count,  and  that  the  count  be  stated  as  so 
many  pair  per  cc.  Linear  groupings  of  three  or  more  coccus  forms  are  to 
be  counted  as  streptococci,  and  the  count  is  to  be  stated  as  so  many  chains 
per  cc. 

B.  That  sterile  evaporated  milk  be  declared  below  standard  on  the 
following  counts: 

100,000,000,  or  more,  pair  of  diplococci  and  diplobacilli  per  cc.,  or 

300,000,  or  more,  chains  of  streptococci  per  cc.,  or 

both;  with  or  without  the  presence  of  body  cells  or  any  considerable  amount  of  organic 
debris,  and  with  or  without  bacilli,  tetracocci,  or  other  associated  microorganisms. 

C.  That  in  case  of  non-sterile  evaporated  milk,  or  raw  milk  intended 
for  the  canning  and  evaporating  process,  and  in  inadequately  processed 
canned  milk,  or  milk  in  imperfectly  sealed  tins,  or  milk  in  unsuitable  tins, 
(tins  with  "Friction  caps,"  for  example),  the  direct  microscopical  ex- 
amination be  supplemented  by  the  usual  plating,  tube,  and  fermenta- 
tion tube  culture  methods. 

D.  That  under  organic  debris  there  should  be  included  such  organic 
particles  as  are  distinctly  recognizable  as  other  than  sugar  crystals, 
casein  granules  or  casein  masses,  or  particles  of  butter  fat.     It  may  in- 
clude the  following  substances: 

a.  Vegetable  tissue  elements  derived  from  field,  soil,  stable  manure,  or  cattle  feed. 

b.  Dirt  particles. 

c.  Stringy  shreds  of  albuminous  matter. 

d.  Variously  colored  (mostly  reddish  brown),  irregular,  amorphous,  or  somewhat 
crystalline  particles  of  resinoid  character,  probably  largely  of  vegetable  origin  (decom- 
position products). 

e.  More  or  less  disintegrated  and  not  distinctly  recognizable  body  cells  (epithelium, 
leucocytes,  endothelial  cells). 

These,  and  other  particles  of  organic  debris,  are  readily  observable 
under  the  low  power  of  the  compound  microscope.  In  homogenized 
milks  that  streptococcus  chains  are  often  well  broken  up,  becoming  largely 
reduced  to  diplococcus  forms  and  also  to  coccus  forms. 

In  the  examination  of  full  milks,  fresh,  evaporated,  sweetened  or 
unsweetened,  and  inclusive  of  creams,  ice  creams,  etc.,  a  small  high  speed 


BACTERIA   IN    THE    INDUSTRIES  1 99 

(2800  revolutions  per  minute)  hand  centrifuge  is  required.  Ice  creams, 
creams,  sweetened  evaporated  milks,  must  be  suitably  diluted  (1-5  as  a 
rule)  before  centrifuging  in  order  to  facilitate  the  separation  of  the  bacteria 
and  organic  debris  and  the  butter  fat.  In  case  of  sweetened  evaporated 
milks,  the  dilution  must  be  sufficient  to  dissolve  all  of  the  lactose  and 
added  sucrose.  To  hasten  the  solution  of  the  lactose,  heat  may 
be  employed. 

Place  10  cc.  of  the  milk  or  the  dilutions  thereof  in  special  two  part 
(i  cc.  +  9  cc.)  tubes  and  centrifuge  for  ten  minutes.  Remove  the  i  cc. 
end  tube,  holding  the  sediment,  and  mix  the  contents  thoroughly  and 
from  this  make  the  bacterial  counts,  using  dilutions  as  may  be  required. 

The  coccus  forms  and  other  microorganisms  in  heat  sterilized  milks 
have  lost  much  of  their  original  staining  properties.  Dead  bacteria 
generally,  especially  those  killed  by  moist  heat,  as  a  rule,  react  feebly  with 
the  usual  stains.  The  streptococcus  form  found  in  evaporated  canned 
milk  most  generally  seen,  appears  to  have  the  morphological  characteristics 
of  the  Streptococcus  acidi  lactici  of  Kruse,  which  is  believed  to  be  entirely 
harmless  to  man  and  to  be  in  normal  lactic  acid  fermentation  of  milk. 
It  is,  however,  quite  immaterial  from  the  standpoint  of  the  purity  and 
freshness  of  milk,  whether  the  organisms  found  are  harmless  or  not. 

In  numerous  samples  of  canned  milk  where  the  coccus  forms  were 
counted,  the  numbers  ranged  from  15,000,000  to  200,000,000  per  cc.  and 
over,  and  the  coccus  count  as  a  rule  exceeded  the  sum  of  the  diplobacillus, 
diplococcus  and  streptococcus  counts  in  the  ratio  of  4:3. 

Based  upon  the  examination  of  numerous  samples  of  several  brands 
of  evaporated  skimmed  milks,  a  count  of  100,000,000  pair  per  cc.  of 
diplo.  forms,  as  a  rule,  corresponds  to  about  150,000,000  coccus  forms, 
per  cc.  or  a  total  of  250,000,000  microorganisms  per  cc.  There  appears  to 
be  no  fixed  number  ratio  between  coccus  and  diplo.  forms  on  the  one  hand 
and  streptos.  on  the  other,  hence  the  suggestion  that  these  groups  be 
given  separate  rating  values. 

4.  Municipal  Milk  Scoring. — In  addition  to  a  score  card  which  indi- 
cates the  condition  of  the  dairying  establishment  and  which  score  will 
indicate  whether  or  not  the  owner  of  the  dairy  is  complying  with  the  re- 
•quirements  of  the  city  ordinance,  the  laboratory  analysts  are  required  to 
make  examinations  and  fill  out  report  blanks,  after  the  following. 

A.  TABLE  FOR  SCORING  CHEMICAL    EXAMINATIONS  OF  MILK 

Total  solids;  Score  100 

13.2  %  or  more 

For  each  .1  %  under  13.2  %  deduct  2  points  on  score. 
For  added  water,  deduct  loo  points. 

If  per  cent,  of  butter  fat  is  less  than  3  %,  deduct  100  points. 
For  milk  containing  preservatives,  deduct  100  points. 


200  PHARMACEUTICAL  BACTERIOLOGY 

B.  TABLE  FOR  SCORING  BACTERIOLOGICAL  EXAMINATIONS  OF  MILK 


Raw  Milk 

Pasteurized  Milk 

Grade  A 

Grade  A 

Grade  B 

Bacteria  in 
thousands 
per  c.c. 

Score 
in 
points 

Before  pasteuri- 
zation.   Bac- 
teria in  thou- 
sands per  cc. 

After  pasteuri- 
zation.    Bac- 
teria in  thou- 
sands per  cc. 

Before  pasteuri- 
zation.    Bac- 
teria in  thou- 
sands iper  cc. 

After  pasteuri- 
zation.    Bac- 
teria in  thou- 
sands per  cc. 

Score 
in 
points 

1       • 

50  to  100 

140 

100  to  200 

8  to  10 

500  to  1000 

40  to  50 

70 

25  to    50 

160 

50  to  100 

6  to    8 

400  to    500 

30  to  40 

80 

10  to    25 

180 

?-5  to    50 

4  to    6 

300  to    400 

20  tO  30 

90 

under  10 

200 

under  25 

under  4 

under    300 

under  20 

TOO 

Score  ior  raw  milk  is  obtained  directly  from  table. 

Score  for  pasteurized  milk  is  obtained  by  adding  together  the  score  before  pasteurizing  and  the  score 
after  pasteurizing. 

For  raw  milk  deduct  20  points  for  the  first  1000  colonies  of  the  colon  group  or  streptococci,  whichever 
may  be  the  more  numerous,  and  deduct  10  points  for  each  subsequent  1000. 

For  milk  after  pasteurization  deduct  10  points  for  the  first  100  colonies  of  the  colon  group  or  strepto- 
cocci, whichever  may  be  the  more  mumerous,  and  deduct  2  points  for  each  subsequent  100. 

4.    THE  LACTIC  ACID  MICROBE  AND  KEFIR  PREPARATION 

Within  recent  years  the  subject  of  intestinal  digestion  and  the  relation- 
ship of  intestinal  microbes  to  digestion  and  longevity  has  received  much 
attention.  Metchnikoff  declares  that  the  early  senile  cell  changes  in  the 
body  are  due  to  the  repeated  or  chronic  autointoxications  brought  about 
by  certain  noxious  intestinal  ferments  of  bacterial  origin  which  are  absorbed 
into  the  circulation.  Some  of  these  bacteria,  especially  those  found  in  the 
small  intestines,  are  beneficial,  secreting  enzymes  which  aid  digestion,  but 
the  enormous  quantity  of  microbes  active  in  the  lower  large  intestine  are 
for  the  most  part  injurious,  producing  putrefactive  changes,  liberating 
toxins  which  when  absorbed  into  the  system  in  sufficient  quantity  produce 
the  symptoms  of  ptomaine  poisoning. 

In  order  to  combat  these  objectionable  bacterial  activities,  it  is  neces- 
sary to  regulate  the  bacterial  development  in  the  large  intestine.  Lactic 
acid  has  long  been  known  as  an  efficient  remedy  in  the  treatment  of  various 
intestinal  disorders.  It  is  known  that  the  poor  of  certain  European 
countries  who  live  largely  on  potatoes  and  clabbered  or  thick  milk  are 
notably  free  from  intestinal  disorders  and  are  remarkably  long-lived.  It 
is  known  that  pickles,  sauerkraut  and  sour  milk  are  excellent  bowel 
regulators,  in  spite  of  the  fact  that  these  foods,  the  former  two  in  particular 
are  well-nigh  indigestible  and  have  little  food  value.  The  Arabians  have 
long  used  koumys  as  a  healthful,  life-prolonging  article  of  diet.  To  this 
class  of  foods  also  belongs  the  Bulgarian  yoghurt  and  the  Egyptian  raib. 

The  ferments  of  koumys,  kefir,  yoghurt  and  raib  resemble  each  other  in 


BACTERIA   IN    THE    INDUSTRIES  2OI 

that  they  are  mixed,  consisting  of  several  lactic-acid  microbes  or  organisms 
and  yeast  organisms.  These  foods  or  drinks  therefore  contain  lactic  acid 
and  a  small  amount  of  alcohol. 

As  soon  as  it  was  determined  experimentally  that  the  beneficent  action 
of  sour  milk,  thick  or  clabbered  milk  and  the  above-named  special  prepara- 
tions was  largely  due  to  the  lactic  acid  formed  by  specific  microbes,  efforts 
were  made  to  isolate  these  organisms  in  pure  culture  and  to  induce  them 
to  act  in  sterile  or  pure  milk.  This  has  been  done,  and  there  are  now  upon 
the  European  and  American  market  several  patented  preparations  con- 
sisting of  the  lactic  acid  bacillus. 

Our  knowledge  of  the  relative  importance  of  the  several  organisms 
which  are  said  to  produce  the  fermentative  changes  in  the  milk  is  as  yet 
incomplete.  Bacteriologists  have  thus  far  not  succeeded  in  disclosing 
all  of  nature's  secret  processes  involved.  It  is  supposed  that  the  microbe 
of  Bulgarian  sour  milk,  the  Bacillus  bulgaricus,  is  the  most  vigorous  and 
active  of  all  organisms  concerned  in  the  lactic-acid  fermentation  of  milk. 

It  is  not  definitely  determined  whether  or  not  the  fermentations  of 
milk  induced  by  the  mixed  and  often  filthy  "yeasts"  employed  in 
making  koumys,  kefir,  yoghurt,  matzoon  and  other  similar  fermented 
foods,  are  superior  or  inferior  to  those  of  lactone  and  other  pure 
culture  milk  ferments.  It  is,  however,  very  evident  that  the  mar- 
keted preparations  in  tablet  form  give  very  satisfactory  results,  as 
used  by  pharmacists  and  in  the  home.  Full  directions  for  using 
the  tablets  are  found  on  every  package.  As  is  naturally  to  be  sup- 
posed, these  tablets  deteriorate  in  a  comparatively  short  time  and  all 
reliable  manufacturers  place  the  age-limit  on  each  package. 

Pharmacists  can  prepare  a  marketable  kefir  ferment  powder  from  milk 
activated  by  kefir,  provided  care  is  observed  to  guard  against  outside 
infection  in  the  several  steps  of  procedure.  The  following  is  the  method 
of  preparing  a  kefir  powder: 

A.  Securing  the  Kefir. — The  kefir  known  as  kefir  grains  or  kefir  seeds 
may  be  secured  from  the  large  dealers  in  drugs  in  New  York  City  or  in 
other  large  Eastern  port  cities.     The  kefir  is  a  solid  of  a  tough  gelatinous 
consistency,  brittle  when  dry,  of  grayish-yellow  color.     It  is  a  conglomera- 
tion of  various  organisms,  as  Dispora  caucasica,  several  species  of  other 
microbes,  a  yeast  organism,  and  other  undetermined  organisms. 

B.  Washing  the  Kefir. — Place  two  or 'three  drams  of  the  kefir  in  a  mix- 
ture of  equal  parts  of  milk  and  water,  enough  to  cover  the  kefir.     Allow 
to  stand  for  four  hours,  decant  off  the  liquid  and  renew  at  intervals  of 
about  one  hour.     Repeat  this  four  or  five  times  at  a  temperature  of  about 
82°  F.     I 

This  process  serves  a  cleansing  purpose  and  initiates  the  fermentative 


2O2  PHARMACEUTICAL  BACTERIOLOGY 

change.     The  amount  used  will  depend  upon  the  quantity  of  powder  to  be 
made. 

C.  Preparing  the  New  Kefir. — -Wrap  the  washed  and  softened  kefir  in  a 
piece  of  sterilized  gauze  and  place  it  in  one  quart  of  pasteurized  milk. 
Keep  at  'a  temperature  of  82°  F.     Allow  to  stand  for  from  twelve  to  fifteen 
hours,  until  the  milk  is  curdled. 

D.  Skimming  and  Draining  the  Kefir. — Remove  the  cream  and  drain 
the  curd  (kefir)  in  sterilized  gauze  until  quite  dry. 

E.  Drying. — -Add  (to  the  drained  kefirized  curd)  an  equal  weight  of 
sugar  of  milk,  mix,  and  spread  thinly  upon  sterilized  gauze  or  upon  a  sterile 
glass  plate  and  dry  in  a  current  of  sterile  warm  air  (80°  F.). 

F.  Powdering. — Powder  the  dried  mass  gently  and  put  up  in  dry,  sterile, 
one-ounce,  wide-mouthed  vials,  closed  with  sterilized  corks. 

G.  Directions  for  Use* — Upon  the  bottles  place  the  following  directions 
for  using  the  powder  thus  prepared:  "Dilute  one  quart  of  milk  with  one- 
half  pint  of  water,  add  a  pinch  of  salt  and  one  level  teaspoonful  of  the 
powder.     Set  aside  for  twelve  to  fifteen  hours  at  a  temperature  of  85°  F. 
shaking  frequently.     Use  at  once  or  keep  on  ice." 

There  are,  of  course,  no  conveniences  for  regulating  the  temperature  in 
the  average  household,  and  the  action  of  the  powder  must  take  place  at  the 
ordinary  temperature  of  the  home.  Thus  the  time  required  to  curdle  the 
milk  will  vary.  The  powder  should  be  kept  in  a  cool  or  cold,  dry  place. 
Of  course,  a  small  amount  of  kefirized  milk  can  be  used  to  curdle  any 
quantity  of  fresh  milk  without  using  any  of  the  powder. 

The  pharmacist  should  test  the  kefir  which  he  is  about  to  use  in  pre- 
paring the  powder,  in  order  to  be  certain  that  it  is  active  in  curdling  milk. 
Likewise  should  he  test  the  powder  prepared  from  it. 

The  kefir  powder  above  described  is  similar  to,  although  not  identical 
with,  certain  microbic  lactic-acid  ferments  found  on  the  market,  as  the 
lactone  tablets,  bacillary  tablets,  yoghurt  tablets,  fermenlactyl,  lacto- 
bacilline  and  others.  These  are  prepared  from  pure  cultures  of  species  of 
lactic-acid  bacilli,  dried  and  formed  into  tablets  with  some  pulverulent 
(starch,  milk,  sugar)  base,  ready  for  use.  The  milk  (in  quart  bottles)  is 
first  pasteurized,  a  pinch  of  salt  is  added  and  two  or  three  tablets  are  crushed 
and  mixed  with  the  milk.  In  a  day  or  so  the  milk  is  transformed  into  an 
acidulous  drink,  resembling  buttermilk  somewhat  in  flavor,  though  it  is 
not  buttermilk,  as  is  generally  supposed. 

These  tablets  have  gained  in  favor  within  recent  years.  They  deterio- 
rate in  time,  as  already  stated,  and  the  time-limit  is  stamped  on  each 
container.  Like  the  kefir,  they  act  more  quickly  at  a  temperature  of 
about  25°  C. 

As  may  be  readily  understood,  kefir, lactone,  etc.,  will  not  produce  the 


BACTERIA   IN    THE    INDUSTRIES  203 

characteristic  changes  in  milk  to  which  preservatives  have  been  added ;  in 
fact,  the  failure  to  produce  fermentation  is  an  indication  that  preservatives 
are  present. 

5.  MICROBIC  PEST  EXTERMINATORS 

Attempts  have  been  made  from  time  to  time  to  exterminate  certain 
animal  pests  by  inoculating  them  with  some  fatal  contagious  disease  of 
microbic  origin.  Experiments  along  this  line  have  been  carried  on  for 
some  time,  ever  since  the  causative  relationship  of  microbes  and  disease 
was  fully  established;  but  it  is  only  within  recent  years  that  extensive 
practical  application  was  made  of  the  use  of  a  few  microbic  pest  extermina- 
tors. One  of  the  first  to  be  used  with  some  success  was  the  chintz-bug 
exterminator.  The  chintz-bug  (Blissus  leucopteris,  also  called  chinch-bug 
chink-bug)  was  a  very  destructive  corn  (Zea  mays)  pest  of  the  Central 
States  (Illinois,  Kansas,  Nebraska,  Iowa) ,  causing  great  damage  to  crops 
during  certain  very  dry  seasons.  Extensive  experiments  carried  on  at 
the  University  of  Illinois  and  also  at  the  University  of  Minnesota  (Depart- 
ments of  Agriculture)  led  to  the  discovery  of  a  microbic  disease  of  this  pest 
which  was  quickly  fatal  and  which  spread  very  rapidly.  The  insects,  in 
cages,  were  inoculated  with  pure  cultures  of  the  pathogenic  microbes,  and 
insects  in  the  diseased  condition  were  sent  to  the  farmers  with  instructions 
how  to  scatter  them  through  an  infested  corn-field.  The  results  were  in 
some  instances  very  satisfactory,  and  again  without  appreciable  effects. 
The  trouble  in  the  use  of  this  exterminator  lay  in  the  fact  that  the  climatic 
conditions  (rainy,  damp  weather)  essential  to  the  spreading  of  the  disease 
did  not  generally  prevail,  and  as  soon  as  the  climatic  conditions  were 
favorable  inoculation  became  unnecessary,  as  the  disease  developed 
without  artificial  aid  and  effectuallv  checked  further  ravages. 

Rabbits  are  one  of  the  very  annoying  field  pests  of  Australia,  and  at- 
tempts have  been  made  to  exterminate  them  by  means  of  pure  cultures  of 
microbes  capable  of  developing  a  fatal  infectious  disease  among  these 
animals,  but  the  results  were  quite  unsatisfactory. 

More  recently  there  have  been  placed  on  the  market  quite  an  array  of 
mice  and  rat  exterminators  of  microbic  origin  under  various  trade  names 
as  ratin,  rat  virus,  azoa,  rattite,  Danysz  virus  and  mouratus.  These  pre- 
parations consist  of  pure  cultures  of  bacilli  pathogenic  to  rats  and  mice, 
as  the  Bacillus  murisepticus  and  Bacillus  typhimurium,  mixed  with  some 
inert  base,  as  corn-meal,  oat-meal,  etc.,  forming  a  coarse  powder.  Some 
preparations  are  in  liquid  form.  They  are  used  by  mixing  the  powder  or 
liquid  with  moist  corn-meal  or  other  food  material  relished  by  these 
animals,  and  spreading  it  near  their  haunts  and  runs.  Fortunately, 
these  substances  are  harmless  to  man  and  animals  other  than  mice  and 


2O4  PHARMACEUTICAL  BACTERIOLOGY 

rats.  These  microbic  rat  and  mice  exterminators  have  thus  far  proven 
to  be  rather  unsatisfactory.  They  have  undoubtedly  given  excellent 
results  in  some  instances,  and  again  they  have  been  absolute  failures. 
The  tests  made  by  the  University  of  California,  and  by  Dr.  Rupert  Blue 
in  his  famous  plague-rat  extermination  in  San  Francisco,  have  given 
almost  wholly  negative  •  results.  A  microbic  squirrel  exterminator 
("squirrelin")  has  proven  entirely  unsatisfactory. 

When  we  consider  how  difficult  it  is  to  prevent  fatal  epidemics,  it  cer- 
tainly does  seem  reasonable  to  suppose  that  it  should  be  a  comparatively 
easy  matter  to  find  ways  and  means  for  disseminating  fatal  epidemics,  but 
so  far  the  commercial  attempts  made  in  that  direction  have  proven  rather 
discouraging.  Further  carefully  conducted  experiments  along  this  line 
are  necessary.  It  is  known  that  the  ravages  of  certain  pests  are  sometimes 
suddenly  checked  by  the  natural  invasion  of  some  pathogenic  organisms. 
This  is  frequently  observed  among  plant  lice  (Aphis)  and  other  insect 
enemies  of  plants. 

6.  BACTERIA  IN  THE  TANNING  INDUSTRY 

i '  .  The  object  in  tanning  leather  is  to  protect  it  against  decomposition  and 
to  render  it  pliable.  The  various  animal  hides  reaching  the  tannery 
are  preserved  by  drying  and  salting.  At  the  tannery  the  hides  are  treated 
as  follows : 

A.  Removing  the  Hair —Depilation.— This  is  done  by  means  of  chemi- 
cals, as  lime  or  sodium  sulphite,  or  through  the  agency  of  rotting  bacteria, 
as  Bacillus  (Proteus)  vulgaris  and  others.    Just  which  of  several  species 
of- rotting  bacteria  is  most  active  in  this  process  has  not  been  definitely 
determined. 

B.  Drenching  or  Bating. — After  the  hair  has  been  removed,  the  hides 
are  macerated  in  an  aqueous  solution  of  the  excrement  or  dung  of  pigeons, 
hens  and  dogs.    These  substances  set  up  a  lactic  acid  fermentation  due  to 
the  microbes  contained  therein.     The  active  organisms  have  not  been  iso- 
lated as  yet;  Bacillus  gasoformans  and  B.  erodiens  are  perhaps  active,  but 
there  are  also  present  many  yeasts,  moulds  and  other  organisms  which  may 
have  their  special  effects. 

The  first  part  of  this  process,  known  as  "  bating,"  is  initated  by  bird 
dung;  the  second  process,  known  as  "puring,"  is  due  to  the  action  of 
dog  dung.  Attempts  have  been  made  to  use  pure  cultures  of  the  active 
microbes  to  supplant  these  filth  substances,  but  so  far  these  efforts  have 
not  proven  wholly  successful. 

C.  Tanning.— The  bated  hides  are  next  treated  in  the  tan  pit  (coarse 
sMris)  'or  in  bark  liquor  (soft  thin  skins),  where  the  souring  process  takes 

r 'This  process  is  also  due  to  bacterial  activity.     Our  knowledge  of 


BACTERIA   IN    THE   INDUSTRIES  205 

the  action  which  takes  place  and  of  the  bacteria  involved  is  very 
incomplete. 

Bacteria  are  important  factors  in  siloing;  in  curing  tobacco^  tea  and 
cacao.  The  flavor  of  different  brands  of  tobacco  is  due  to  different  bac- 
teria, and  attempts  have  been  made  to  isolate  those  producing  desirable 
flavors  and  to  use  them  in  pure  culture.  It  is  highly  probable  that  the 
bouquet  of  old  wines  is  due  to  bacterial  action.  These  are,  however, 
matters  which  require  further  study.  Rotting  bacteria  are  active  in 
paper-making.  In  the  maceration  process  certain  bacteria  feed  upon  and 
decompose  the  less  resisting  vegetable  cell-walls,  as  those  of  the  paren- 
chyma tous  tissues,  the  epidermal  tissue,  etc.,  leaving  the  more  resisting 
fibrous  lignified  tissues  as  bast  and  wood  fibers.  The  pulp  is  then  poured 
on  sieves  and  the  rotted  or  digested  portions  washed  out. 

Bacteria  are  now  practically  employed  in  the  purification  of  sewage. 
This  is  done  in  what  are  known  as  "contact  beds,"  in  which  the  environ- 
ment is  made  favorable  *to  rapid  development  of  those  non-pathogenic 
rotting  bacteria  which  disintegrate  the  organic  substances  and  at  the  same 
time  prevent  the  development  of  the  pathogenic  or  otherwise  objectionable 
microbes.  It  is  highly  probable  that  this  method  may  be  applied  to  the 
purification  of  streams  and  other  large  bodies  of  water. 

The  possibilities  in  the  practical  utilization  of  bacteria  in  the  arts  and 
industries  are  promising,  and  it  may  confidently  be  expected  that  wonder- 
ful innovations  along  this  line  will  be  made  in  the  very  near  future. 

7.  MICROBIOLOGICAL  METHODS  IN  FOOD,  MILK  AND  WATER 

ANALYSIS 

It  is  not  the  province  of  a  work  of  this  kind  to  enter  into  the  discussion 
of  special  methods  of  analysis.  These  must  be  gotten  through  other 
sources  and  channels.  The  following  practical  methods  are  given  for  the 
benefit  of  those  who  are  endowed  with  sufficient  inherent  ability  to  apply 
them. 

i.  Water  Analysis.  Plankton  Examination. — V.  Hensen  applied  the 
term  plankton  to  the  minute  organisms  and  other  organic  matter  drifting 
or  floating  in  fresh  and  in  salt  water.  The  term  therefore  includes  bac- 
teria and  other  microorganisms,  inclusive  of  dead  organic  matter  and  of 
mineral  matter.  In  the  more  limited  sense  plankton  excludes  bacteria; 
although  there  is  no  satisfactory  reason  why  this  group  should  be  ex- 
cluded. The  following  methods  have  been  used  more  or  less. 

a.  Hassell  Specific  Gravity  Method. — Let  two  or  three  liters  of  the 
water  to  be  examined  stand  in  some  suitable  vessel  for  twelve  to  twenty- 
four  hours.  Decant  all  but  200  cc.  This  remainder  of  200  cc.  with  all 
sediment,  is  poured  into  a  conical  test  glass  with  rounded  bottom 


206  PHARMACEUTICAL  BACTERIOLOGY 

set  aside  for  six  hours.  Pour  oft  the  supernatant  liquid  and  examine 
the  small  amount  of  sedimentary  residue  microscopically.  In  order  to 
reduce  to  a  minimum  the  growth  of  the  contaminating  organisms  during 
the  time  interval  above  indicated,  the  containing  vessels  should  be  placed 
in  an  ice  chest.  The  method  is  satisfactory  as  far  as  the  estimation  of 
inorganic  and  dead  organic  water  contamination  are  concerned,  but  is 
very  unsatisfactory  as  far  as  the  living  contaminations  are  concerned. 

b.  MacDonald  Gravitation  Method. — Into  a  vessel  containing  one  to 
two  liters  of  water,  place  a  watch  crystal  and  let  stand  for  twenty-four 
hours.     Siphon  off  the  water,  carefully  remove  the  watch  crystal  with  the 
sediment  and  examine  microscopically.     The  deposit  in  the  watch  crystal 
represents  the  amount  of  material  derived  from  a  volume  of  water  equal 
to  the  diameter  of  the  watch  crystal  times  the  height  of  the  water  column 
in  the  vessel. 

c.  Kean  Sand  Filtration  Method. — Run  100  cc.  of  water  through  a 
funnel  with  a  sand  plug  (clean  fine  quartz  sand).*   Wash  the  sand  carrying 
the  plankton  into  a  watch  crystal  by  means  of  i  cc.  of  water  and  examine 
microscopically. 

d.  The  Sedgwick-Raf  ter  Method. — This  is  a  further  development  of  the 
Kean  method  and  has  been  quite  extensively  employed  in  the  United 
States.     Sand  of  definite  fineness  is  to  be  used  and  2  50  cc.  of  the  water  are  to 
run  through  the  filter.     A  specially  constructed  counting  chamber  (50  by 
20  by  i  mm.,  hence  holding  i  cc.  of  the  sediment)  is  to  be  used  and  the 
counting  is  to  be  done  by  means  of  a  specially  ruled  micrometer  scale 
and  a  %  inch  objective.     Bacterial  counting  cannot  be  done  according  to 
this  method,  neither  can  the  smaller  algae  and  protozoa  be  accurately 
counted. 

e.  The  Dibdin  Double  Filtration  Method. — Run   i   liter  of  water 
through  filter  paper.     Wash  residue  into  a  clay  filter  made  by  plugging 
the  drawn  out  end  of  a  piece  of  combustion  tubing  with  a  mixture  of  baked 
clay  and  kieselguhr .    Filtration  is  accomplished  by  means  of  suction.    The 
sediment  (plankton)  forms  a  compact  cylindrical  layer  in  the  tube  and  can 
be  measured  and  the  amount  of  the  sediment  per  liter  of  the  water  stated. 
The  sedimentary  plug  can  then  be  removed  and  examined  microscopically. 
This  method  likewise  precludes  the  estimation  of  bacteria  and  also  some 
other  very  minute  organisms. 

f.  Centrifugal  Method. — Centrifuge  ten  to  fifty  cc.  quantities  of  the 
water  to  be  examined  at  a  high  rate  of  speed,  so  as  to  throw  down  all  matter 
in  suspension,  inclusive  of  bacteria.    Decant  or  pipette  off  all  but  about 
i  cc.  of  the  water,  make  up  to  two  or  three  cc.  by  adding  filtered  distilled 
water,  shake  or  mix  thoroughly  and  make  the  counts  by  means  of  a  suitable 
counting  chamber.     Should  actively  motile  organisms  such  as  paramecia, 


BACTERIA   IN   THE   INDUSTRIES  207 

interfere  with  the  counting,  they  may  be  killed  by  adding  a  drop  or  two  of 
ether  or  chloroform,  to  the  centrifuged  material.  For  the  purpose  of 
examining  and  comparing  a  number  of  water  samples  at  one  operation, 
the  Stewart-Slack  centrifuge  head  will  be  found  very  convenient  and  time 
saving. 

2.  Milk  Analysis. — There  are  three  methods  of  direct  examination 
of  milk  in  use.     Two  of  these  require  the  use  of  the  centrifuge.     The 
direct  microscopical  examination  of  milk  will  convey  information  as  to 
quality  and  purity  which  cannot  be  obtained  through  any  of  the  chemical 
methods. 

a.  The  Stewart  Slack-Method. — Two  cc.  of  milk  are  placed  into  glass 
tubes  which  are  then  closed  at  both  ends  by  means  of  suitable  rubber 
stoppers.     The  special  centrifuge  heads  made  according  to  the  specifica- 
tions of  Dr.  Stewart  of  the  Philadelphia  Board  of  Health  will  hold  twelve 
such  tubes.    The  tubes  are  then  centrifuged  at  2,000  to  3,000  revolutions 
per  minute,  for  ten  minutes.     The  sediment  which  has  become  more  or  less 
fixed  upon  the  stopper  in  the  lower  end  of  the  tube  is  mixed  with  a  drop 
or  two  of  water  and  smeared  upon  a  slide,  so  as  to  cover  about  four  square 
centimeters  of  space,  allowed  to  dry  and  stained  with  methylene  blue. 
The  examination  is  made  with  the  oil  immersion  and  bacteria,  pus  cells 
and  epithelial  cells  counted. 

b.  The  Doane-Buckley  Method. — Ten  cc.  of  the  milk  is  centrifuged 
for  four  minutes  at  a  speed  of  2,000  revolutions  per  minute.     The  fat  is 
carefully  removed;  centrifuged  for  one  minute  more  and  the  fat  again 
carefully  wiped  away  by  means  of  a  small  cotton  swab.    Pipette  off  the 
supernatant  fat  free  milk  and  mix  the  sediment  with  two  drops  of  a  satu- 
rated alcoholic  solution  of  methylene  blue.     Let  the  stain  act  for  a  few 
minutes,  assisted  by  warmth.     Make  up  to  the  i  cc.  mark  of  the  tube  by 
adding  water.     Examine  for  body  cells  by  means  of  the  hemacytometer. 

c.  The    Prescott-Breed  Method. — Moo   cc'   °f  a  thoroughly  mixed 
sample  of  the  milk  is  spread  on  a  slide,  within  the  ruled  lines  of  just 
i  square  cm.     The  >foo  cc-  of  milk  is  measured  by  means  of  a  special 
graduated  capillary  tube.     Both  the  pipettes  and  the  ruled  slides  are  to 
be  had  in  the  market.     However,  both  can  be  made  in  the  laboratory  by 
anyone  with  ordinary  mechanical  skill.     The  slide  with  the  milk  smear 
is  set  aside  to  air  dry,  fixed  with  alcohol  and  then  stained  with  methylene 
blue  or  some  other  blood  stain.     The  counting  is  done  with  the  oil  immer- 
sion, after  having  determined  the  area  covered  by  the  field  of  view. 
Estimations  are  made  of  the  number  of  bacteria  and  body  cells  per  cc. 
of  the  milk. 

3.  Examination    of    Tomato    Products. — The    industry    of   canning 
tomatoes  has  reached  enormous  proportions  in  the  United  States  within 


208  PHARMACEUTICAL  BACTERIOLOGY 

recent  years.  Much  of  the  material  placed  in  cans  and  offered  in  the 
market  is  of  very  inferior  quality,  being  made  from  tomato  refuse,  trim- 
mings and  rotten  tomatoes.  Two  methods  of  direct  microscopical  exam- 
ination of  these  products  are  in  use. 

a.  The  Howard  or  Bureau  of  Chemistry  Method. — The  method  con- 
sists of  three  parts,  as  follows : 

Mold  Counting. — A  bit  of  the  tomato  product  (as  catsup,  puree,  sauce, 
paste)  is  placed  on  the  special  Howard  mold  chamber  (depth  34o  mm.) 
and  covered  with  a  thick  cover  glass.  The  counting  is  done  by  means  of  a 
low  power  objective,  the  draw  tube  being  so  adjusted  that  the  diameter 
of  the  field  of  view  is  just  exactly  1.83  mm.  (therefore  an  area  of  about 
2.5  sq.  mm.).  Each  field  showing  mold  hyphae  extending  %  of  the  way 
across,  or  more,  is  counted  as  positive  mold.  A  tomato  product  is  con- 
sidered unsuitable  for  human  consumption,  if  it  shows  66,  or  more,  per  cent, 
of  the  fields  of  positive  mold. 

Spore  Counting. — The  spore  count  includes  mold  spores  of  all  kinds 
as  well  as  yeast  cells  and  the  counts  are  recorded  as  so  many  per  J^o 
cubic  mm. 

Bacterial  Counting. — The  hemacytometer  is  used.  Only  the  large 
"rod  shaped  forms  "are  counted,  it  not  being  specified  whether  the 
smaller  forms  and  the  diplobacilli  are  to  be  included. 

The  Howard  method  has  been  severely  criticized  for  several  reasons. 
It  does  not  allow  for  differences  in  the  consistency  of  the  various  tomato 
products.  The  fact  that  only  "rod  shaped"  bacteria  are  counted  has 
rendered  the  bacterial  counting  practically  valueless  as  far  as  the  protec- 
tion of  the  consumer  is  concerned,  since  the  distinctively  rod  shaped  bac- 
teria found  in  tomato  products  are  mostly  of  the  lactic  acid  forming  group, 
and  largely  harmless,  whereas  the  coccus  forms,  streptococcus  forms,  small 
diplobacillus  forms,  to  which  groups  belong  many  of  the  filth  and  sewage 
types,  are  not  counted.  There  is  also  no  excuse  for  the  unusual  fractional 
recording  of  the  spores  and  there  should  be  a  distinction  made  between 
spores  and  yeast  cells.  In  those  laboratories  not  under  the  direction  of 
Federal  and  State  pure  food  law  administration,  the  Howard  method  is 
modified  so  as  to  conform  to  other  more  scientific  methods  of  foods  exam- 
ination. The  defenders  of  the  Howard  method  declare  that  the  method  is 
purposely  simplified  in  order  to  suit  it  to  the  capabilities  of  the  analysts 
who  are  employed  to  do  the  microscopical  work  in  the  various  laboratories. 
This  argument  is  not  worthy  of  serious  consideration.  Incompetent 
analysts  have  no  place  in  any  laboratory,  least  of  all  in  a  laboratory  where 
matters  affecting  the  public  health  are  concerned. 

4.  General  Method  for  Making  Direct  Bacterial  Counts. — The  follow- 
ing method  is  practically  applicable  in  the  direct  microscopical  examina- 


BACTERIA   IN    THE    INDUSTRIES  2OQ 

tion  of  food  substances  of  all  kinds.  Weigh  or  measure  a  definite  amount 
of  a  well  mixed  average  sample,  mix,  grind  or  triturate  as  may  be  necessary, 
dilute  as  may  be  desired  (1-5,  i-io,  i-ioo),and  make  the  counts  by  means 
of  the  hemacytometer.  If  the  material  is  to  be  stained  in  order 'to  make 
bacterial  .counting  possible,  then  place  J/fo  cc-  of  the  prepared  and  di- 
luted material  upon  a  clean  slide  and  spread  it  out  over  an  area  of  10  sq. 
cm.  (2  cm.  by  5  cm.),  air  dry,  add  alcohol,  stain  and  make  the  counts  by 
means  of  the  oil  immersion  objective,  without  the  hemacytometer.  The 
pipette  must  have  a  free  delivery  and  must  be  carefully  graduated  into 
tenths  of  one  cc.  This  is  essentially  a  modification  of  the  Prescott  and 
methods  as  applied  to  the  direct  milk  count,  differing  in  that  larger  amounts 
are  used  (Ho  cc-  instead  of  Moo  CC0-  It  is  impracticable  to  use  the 
smaller  amount  in  most  cases,  and  by  using  the  larger  amounts  the  source 
of  error  is  correspondingly  less.  The  area  of  the  field  of  view  with  the 
oil  immersion  lens  must  be  carefully  determined.  We  will  suppose 
that  the  field  of  view  is  J£o  sq.  mm.,  the  dilution  used  i-ioo,  and  the 
average  number  of  bacteria  in  one  field  of  view  is  thirty,  then  the  total 
number  of  bacteria  per  cc.  would  be  1,500,000,000  (50  X  30  X  100  X 
1,000  X  10  =  1,500,000,000). 

Those  interested  in  a  fuller  discussion  of  the  decomposition  changes 
in  food  substances  and  the  microanalytical  methods  employed  in  the 
examination  of  food  substances  should  consult  The  Microbiology  and 
Microanalysis  of  Foods  (P.  Blakiston's  Son  and  Company,  1920.) 


14 


CHAPTER  IX 
ZYMOLOGY— FERMENTS  AND  FERMENTATIONS 

1.  Introduction. — The  terms  ferment  and  enzyme  are  synonymous. 
Occasionally  the  expression  " catalytic  agent"  is  used.     Unfortunately 
we  are  as  yet  very  much  in  the  dark  as  to  the  physical  and  chemical 
nature  of  ferments  or  enzymes  as  well  as  the  processes  comprehended 
under  the  term  fermentation.     The  literature  on  the  subject  is  dis- 
couragingly  voluminous  and  correspondingly  Jacking  in  clearness  and 
conclusiveness.     Of  the  comparatively  recent  works,  that  by    Oppen- 
heimer1  is  the  clearest  and  in  many  respects  the  most  complete.     In  the 
foUowing  presentation  of  this  subject  we  have  followed  this  author  quite 
closely. 

2.  Historical. — As  comprehended  by  the  ancients  fermentation  meant 
a  "boiling"  without  fire,  a  " bubbling"  a  disturbance  in  organic  compounds 
resulting  in  a  marked  change  in  the  appearance  of  the  substance  affected. 
Originally  the  term  applied  almost  wholly  to  the  activities  of  the  yeast 
organisms.     Alcoholic  fermentation  was  known  to  the  ancient  Hindus, 
Arabians,    Greeks  and  Romans.     Centuries  prior  to  the  Christian  era 
the  Goths,  Franks  and  Teutons  made  fermented  drinks  from  grain  (beer) 
and  honey  (mead). 

It  is  noteworthy  that  no  attempts  were  made  to  explain  fermentation 
until  comparatively  recent  times.  Valentinus  (of  Erfurt),  as  late  as  the 
fifteenth  century,  was  among  the  first  to  offer  an  explanation,  stating  that 
it  was  a  process  of  purification,  probably  getting  the  idea  from  the  fact 
that  in  beer  and  wine  fermentation  the  liquids  become  quite  clear  through 
the  settling  of  the  yeast  as  soon  as  the  process  of  fermentation  is  completed. 
In  fact  not  until  the  eighteenth  century  did  the  subject  receive  any  special 
attention  on  the  part  of  chemists,  biologists  and  physiologists.  At  first 
there  was  a  tendency  to  include  under  fermentation  all  of  the  processes  or 
reactions  accompanied  by  visible  gas  formation  or  bubbling,  and  the 
liberation  of  odoriferous  substances.  Putrefaction  and  fermentation  were 
considered  synonymous.  The  causes  of  fermentation  were  supposed  to  be 
mysterious  vital  forces  or  energies  rendered  active  under  special  conditions 
of  light,  temperature,  air  supply  and  contact  stimuli.  Gradually  distinc- 
tions were  drawn  between  "spirituous"  or  vinous  (alcoholic)  fermentation, 
"sour"  (acid  or  vinegar)  fermentation,  and,  putrefaction.  Stahl,  and 

'CARL  OPPENHEIMER.     Die  Fermente  und  Ihre  Wirkungen.    Leipzig,  1900. 

210 


ZYMOLOGY — FERMENTS  AND  FERMENTATIONS         211 

others,  ascribed  fermentation  to  an  internal  activity  or  motion  of  fer- 
menting substances,  resulting  in  a  splitting  up  of  the  molecules. 

Not  until  the  epoch-making  researches  of  Lavoisier  (1789)  and  those 
of  his  follower  Gay-Lussac  (1815)  did  we  have  any  knowledge  of  the  "part 
played  by  the  element  O  in  fermentations  and  in  other  life  processes. 
Lavoisier  explained  very  clearly  the  familiar  vinous  fermentation  in 
which  sugar  underwent  a  chemical  splitting  process,  resulting  in  the  for- 
mation of  alcohol  and  carbonic  acid  gas.  The  name  of  Liebig  (1865) 
is  most  intimately  associated  with  the  subject  of  fermentation,  as  are 
also  the  names  of  Schwann  (1837)  and  Pasteur  (1857).  Liebig  pro- 
mulgated the  theory,  which  was  soon  generally  accepted,  that  fermenta- 
tion was  a  decomposition  process  of  a  chemical  nature,  which  when  once 
initiated  in  the  fermentable  substance  was  capable  of  being  transmitted 
from  molecule  to  molecule,  until  the  entire  mass  had  undergone  a  change. 
Liebig  insisted  that  the  fermentation  processes  were  entirely  chemical 
but  Dumas,  Schwann,  Pasteur  and  others,  soon  demonstrated  that  this 
was  not  the  case,  that  fermentation  was  induced  by  a  special  organic 
substance,  the  ferment,  which  was  formed  by  living  organisms  and  which 
had  the  power  of  causing  a  special  molecular  disturbance  or  catalytic 
action  in  organic  substances,  resulting  in  the  formation  of  new  compounds. 

Since  Schwann  and  Pasteur,  a  host  of  investigators  have  studied 
fermentation  processes,  in  an  effort  to  determine  the  chemistry,  biology 
and  physiology  of  ferments  or  enzymes.  We  may  mention  a  few  of  the 
leading  investigators,  as  Cagniard-Latour  (1835),  Naegeli  (1879),  Loew, 
Hansen  (1883),  de  Bary,  A.  Mayer,  Hoppe-Seyler,  Hiifner,  Arrhenius, 
Oppenheimer  (1900),  Jorgensen  (1909)  and  others.  Within  recent  years 
the  work  that  has  been  done  on  special  ferments  and  fermentation  processes 
and  on  the  commercial  use  and  application  of  ferments,  has  indeed  as- 
sumed colossal  proportions.  To  merely  prepare  a  review  of  the  workers 
and  their  work  would  require  many  years  of  careful  labor. 

It  is  known  that  organic  substances,  in  fact  all  substances,  gradually 
undergo  a  catalytic  change.  In  the  case  of  minerals  and  rock  formations 
this  change  is  indeed  slow,  whereas  in  organic  substances  the  change  is 
comparatively  rapid.  The  chief  influence  of  ferments  is  to  hasten  the 
catalytic  changes  in  organic  substances.  Therefore,  enzymes  do  not 
initiate  any  catalytic  changes  which  would  not  sooner  or  later  take  place 
without  ferments.  This  fact  has  been  the  cause  of  much  speculation  as  to 
the  intrinsic  properties  of  enzymes  in  their  relationship  to  the  cells  which 
form  them  and  to  the  substances  which  they  are  capable  of  catalyzing. 
The  rate  of  catalysis  in  substances,  even  those  of  an  organic  nature, 
without  the  action  of  ferments  is,  however,  largely  speculation  and  for 
our  present  purpose  does  not  require  further  consideration. 


212  PHARMACEUTICAL  BACTERIOLOGY  y 

3.  General. — From  the  above  it  has  no  doubt  become  evident  that 
the  subject  is  far  from  clear  nevertheless  we  may  submit  certain  propo- 
sitions as  being  more  or  less  conclusive  and  which  will  serve  to  elucidate 
some  of  the  more  or  less  problematical  statements  which  follow. 

a.  As  far  as  is  known,  all  substances  which  may  be  designated  as 
ferments,  are  formed  by  living  plasm  within  living  cells.     Ferments  may 
be  developed  in  single-celled  plants  and  animals  and  in  tissues  and  organs 
of  higher  plants  and  animals. 

There  is  some  dispute  whether  or  not  enzymes  came  into  existence 
prior  to  living  matter.  Troland  and  others  assert  that  certain  autocata- 
lytic  enzymes  or  protoenzymes  came  into  existence  spontaneously  in  the 
remote  geologic  periods  and  that  these  greatly  increased  the  chemical 
changes  so  essential  to  the  creation  of  living  plasm.  There  is  no  way  of 
either  proving  or  disproving  the  idea  and  it  is  a  fact  that  all  growth  activi- 
ties manifest  the  characteristics  of  enzymatic  influence. 

b.  Since  no  ferments  have  as  yet  been  isolated  in  purity,  nothing  is 
known  regarding  their  exact  physical  and  chemical  characters  and  prop- 
^erties.     It  is,  however,  generally  conceded  tSaTjthey  are  organic,  of  an 
albuminoid  nature,  and  chemically  quite  complex.. 

c.  Ferments,   under  favorable   conditions,   are   capable   of  inducing 
chemical  changes  in  organic  substances,  resulting  in  new  compounds  which 
are  always  simpler  in  composition  than  the  mother  substance.     In  induc- 
ing these  changes  the  ferment  itself  does  not  undergo  decomposition. 

d.  To  distinguish  between  organized  and 'unorganized  ferments  is  no 
longer  tenable.     All  ferments,  as  far  as  is  known,  are  organized  in  so  far  as 
they  are  of  living  origin. 

e.  Ferments  and  the  end  products  of  their  activities  are  immediately 
independent  of  the  vital  processes  of  the  cells  that  produce  the  ferments. 
The  ferment  or  enzyme  of  yeast  (zymase),  for  example,  is  not  necessary 
to  the  maintenance  of  the  protoplasmic  activity  of  the  yeast  fermenta- 
tion, as  alcohol  and  carbonic  acid  gas,  as  they  are  not  used  in  the  metabolic 
processes  of  the  yeast  cell.     Nevertheless,  the  ferments  or  enzymes  appear 
to  be  essential  to  the  life  of  the  enzyme  forming  organisms. 

f.  Ferments   are   chemically   unstable.     They   are   checked   in  their 
activity  by  low  temperatures  (10°  to  o°  C.)  and  killed  by  high  tempera- 
tures (45°  to  70°  C.). 

g.  Ferments  are  only  slightly  dialyzable,  but  most  of  them  will  pass 
through  porous  niters  (filter  paper,  porous  clay,  etc.),  under  pressure. 

h.  Ferments  are  precipitated  by  alcohol,  though  not  completely. 
They  are  precipitated  in  proportion  to  the  percentage  strength  of  the 
alcohol.  They  are  soluble  in  water,  in  aqueous  solutions  of  glycerin,  in 
weak  acids  and  alkalies,  and  in  neutral  salt  solutions.  In  a  general  way, 


ZYMOLOGY FERMENTS    AND    FERMENTATIONS  213 

substances  undergoing  precipitation,  carry  with  them  any  ferments  that 
may  be  present. 

i.  Under  ordinary  condition  the  enzymatic  action  of  the  ferment  is  not 
complete.  For  example,  the  zymase  does  not  catalyze  all  of  the  sugar  in 
a  solution  into  alcohol  and  carbonic  acid  gas.  The  process  can,  however, 
be  made  to  proceed  to  completion  by  removing  the  end  products  as  they 
are  formed  (as  may  be  done  by  means  of  a  dialyzable  bag  suspended  in  a 
stream  of  water).  The  reason  why  the  process  is  not  completed  under 
ordinary  conditions  is  because  the  ferment  has  a  synthetic  power,  re- 
combining  the  accumulating  end  products  into  sugar.  The  catalytic 
process  is,  however,  always  much  more  active  than  the  synthetic  process, 
at  least  during  the  earlier  stages  of  the  fermentation.  Gradually  the 
catalytic  process  decreases  until  a  stage  is  reached  where  the  catalytic  and 
synthetic  processes  are  approximately  equalized. 

j.  The  queston  is  often  asked  why  are  the  cells  which  form  the  ferment 
and  the  organs  in  which  they  are  active,  not  digested  or  catalyzed  by  the 
ferment?  While  the  question  is  as  yet  not  definitely  settled,  it  is  highly 
probable  that  the  auto-digestion  of  ferment-producing  cells,  tissues  and 
organs,  is  prevented  by  the  formation  of  anti-ferments  or  anti-bodies,  com- 
parable to  the  anti-bodies  or  anti-toxins  formed  in  cells,  tissues,  and  or- 
gans, to  neutralize  the  toxins  of  disease.  It  is  known,  for  example,  that 
under  certain  pathological  conditions,  localized  digestion  of  stomach  tissue 
may  take  place,  as  in  ulcer.  In  such  cases  the  anti-ferment  is  probably 
non-existent  or  in  some  way  inactivated,  neutralized  or  destroyed. 

k.  In  some  instances  it  is  known  that  the  ferment  or  enzyme  is  formed 
as  the  result  of  a  pro-ferment  or  zymogen,  activated  by  a  second  sub- 
stance. For  example,  pepsin  is  not  formed  in  the  stomach  cells,  but 
rather  in  the  cavity  of  the  stomach  from  the  pepsinogen  which  is  formed 
in  the  mucous  cells  of  the  stomach,  activated  by  the  free  hydrochloric  acid 
present. 

1.  Considered  from  the  standpoint  of  their  relationship  to  the  cells 
which  form  them,  enzymes  may  be  divided  into  three  groups  as  follows: 
a.  Those  which  normally  act  dissociated  from  the  cells  which  form  them, 
as  ptyalin,  pepsin,  rennet,  diastase,  etc.  b.  Those  which  normally  act 
in  association  with  the  cells  that  form  them  but  which  may  be  isolated  and 
will  then  continue  the  fermentation,  as  yeast  ferments;  and  c.  Those 
fernebts  which  thus  far  have  not  been  separated  or  isolated  from  the  cells 
which  form  them,  as  many  of  the  bacterial  enzymes. 

m.  The  smallest  amount  of  enzyme  will  catalyze  as  much  fermentable 
material  as  a  large  amount,  provided  it  is  allowed  to  act  for  the  necessary 
length  of  time.  On  the  other  hand,  it  holds  that  the  rate  of  fermentation 
is  directly  proportioned  to  the  amount  of  enzyme  in  action. 


214  PHARMACEUTICAL  BACTERIOLOGY 

n.  Of  equal  amounts  of  enzyme  isolated,  on  the  one  hand,  and  left  in 
their  natural  environment,  on  the  other  hand,  the  latter  are  by  far  more 
active.  Just  why  this  should  be  is  not  clearly  understood;  the  fact  re- 
mains that  the  enzymic  product  of  manufacture  is  very  frequently  quite 
inactive.  No  doubt  the  methods  of  manufacture  have  a  destructive 
influence  upon  the  enzymes,  or  it  may  be  that  we  have  not  yet  learned  how 
to  isolate  the  enzyme  properly.  Our  knowledge  of  the  action  of  the  pro- 
ferment  of  pepsin  makes  it  clear  that  the  present  methods  of  manufactur- 
ing pepsin  are  defective  in  principle.  The  full  strength  of  active  pepsin  is 
found  in  the  stomach  secretion,  but  not  in  the  stomach  extract  or  pepsin 
of  the  market. 

The  earliest  students  of  ferments  and  of  fermentation  noted  certain 
analogies  between  the  actions  of  chemicals  and  metals  in  certain  states  or 
conditions,  and  ferments,  and  it  is  these  analogies  which  started  the  con- 
troversy as  to  whether  the  enzymatic  processes  were  purely  chemical,  or 
due  to  organic  activity.  The  essential  condition  of  the  process  of  fermen- 
tation is  that  the  catalyzing  or  enzymatic  agent  shall  not  appear  in  the 
end  products  of  fermentation  and  that  it  shall  remain  unchanged  chemi- 
cally. In  the  usual  generation  of  O,  heat  is  applied  to  potassium  chlorate 
mixed  with  MnO2  resulting  in  the  conversion  of  the  chlorate  into  the 
chloride  with  liberation  of  O.  In  this  process  the  MnO2  remains  chemi- 
cally unchanged,  simulating  the  action  of  a  ferment  in  that  it  hastens  or 
accentuates  (aided  by  the  heat)  the  catalyzing  process,  and  does  not  appear 
in  the  end  products. 

Again,  it  is  known  that  metals,  as  platinum  or  silver,  in  a  finely  divided 
state,  will  hasten  catalytic  processes.  This  is  also  true  of  certain  metals 
(gold,  platinum,  silver,  copper,  etc.)  in  the  colloidal  state,  designated  as 
metallic  sols.  The  colloids  or  sols  are  prepared  by  placing  the  metallic 
electrodes  (of  the  metals  names)  into  pure  water  and  passing  an  electric 
current  through  them.  If  the  water  is  not  pure,  or  if  it  is  allowed  to 
become  heated,  the  metal  is  deposited  or  suspended  as  a  cloud,  and  does  not 
form  a  true  colloidal  solution.  The  suspended  and  finely  divided  metal 
particles  can  be  filtered  off,  leaving  the  true  metallic  colloidal  solution. 
Sols  thus  prepared  have  the  power  of  hastening  catalytic  processes  with- 
out themselves  undergoing  any  chemical  change.  According  to  Fischer, 
platinum  sol  will  decompose  (catalyze)  hydrogen  peroxide  with  only 
JlJOOjOOO  milligram  of  platinum  in  ice.  of  water.  Metallic  sols  further 
resemble  true  enzymes  in  that  their  catalytic  action  is  readily  inhibited 
by  a  rise  in  temperature  and  also  in  that  they  are  quite  sensitive  to  the 
actions  of  toxic  agents,  as  arsenic,  strychnine,  etc.  Fischer  suggests  that 
the  decomposition  of  true  ferments  by  heat  is  merely  a  physical  change 
and  that  all  ferments  are  perhaps  colloidal  solutions  and  in  consequence 


ZYMOLOGY — FERMENTS   AND   FERMENTATIONS  215 

exceedingly  liable  to  precipitation  and  inactivation.  The  greater  sensitive- 
ness to  temperatures  of  true  ferments  being  probably  due  to  the  associated 
salts,  for  it  is  known  that  the  sensitiveness  to  heat,  of  colloidal  solutions, 
increases  with  the  amount  of  impurities  added.  It  is  known  tEaTtfae  com- 
paratively purer  ferments  are  more  stable  and  less  sensitive  to  heat  than  are 
those  which  are  comparatively  impure.  This  is  a  fact  well  known  to  manu- 
facturers of  such  commercial  ferments  as  pepsin,  pancreatin,  etc.  The 
catalyzing  activities  of  metals  (finely  divided  and  as  sols)  is  however  not 
limited  to  processes  of  chemical  decomposition.  For  example,  in  the 
commercial  synthetization  of  ammonia  from  free  nitrogen  and  hydrogen, 
acted  upon  by  the  electric  spark  and  subjected  to  pressure  (175  atmos- 
pheres) and  heat  (500°  C.),  the  rate  of  ammonia  production  is  very  materi- 
ally increased  in  the  presence  of  certain  metals  in  the  powdered  form,  as 
uranium  osmium,  mercury,  iron  and  platinum.  The  metals  remain  un- 
changed and  exert  their  catalytic  action  for  an  indefinite  period  of  time. 
It  is  also  known  that  the  catalytic  power  of  different  metals  varies  greatly. 
In  the  ammonia  production  referred  to,  osmium  is  far  more  active  than 
uranium  or  iron.  In  practice  the  metals  giving  a  maximum  yield  in 
proportion  to  their  cost  and  accessibility,  are  used,  rather  than  the  more 
active  but  comparatively  rare  and  expensive  metals. 

There  are  many  phenomena  which  are  as  yet  unexplained  and  which 
present  many  of  the  characteristics  of  fermentations.  It  is  probable  that 
many  of  the  life  processes  are  controlled  and  directed  by  enzymes.  The 
influence  of  the  male  reproductive  cell  is  of  such  a  nature.  An  enzyme-like 
substance  perhaps  acts  upon  the  ovum  inducing  indefinite  septation  and 
growth,  resulting  in  a  new  individual.  It  would  appear  that  the  growth 
of  the  body,  of  its  tissues  and  its  organs,  is  directed  by  enzyme-like  stimuli. 

These  growth  enzymes  apparently  occupy  certain  positions  in  the 
body  and  by  their  oxidizing  influence  produce  or  direct  the  various 
complex  chemical  changes  (assimilation)  which  result  in  the  formation 
and  growth  of  the  tissues  and  organs.  Normally  these  growth  ferments 
are  active  in  such  a  manner  (regulated  and  inhibited)  as  to  give  rise 
to  plants  and  animals  which  we  designate  as  normal;  but  occasionally 
there  is  a  disturbance  or  displacement  in  these  enzymes  and  growth 
irregularities  are  the  result,  such  as  local  and  general  dwarfism  and 
giantism,  atavistic  marks  and  anomalies,  supernumerary  fingers  and 
toes,  duplication  of  parts,  twins,  etc.,  etc.  We  may  assume  that  the 
geotropic  position  of  roots  and  stems  and  the  horizontal  position  of 
branches  of  plants,  is  controlled  by  enzymes.  Occasionally  the  nor- 
mal positions  are  reversed  or  cnanged.  It  is  not  uncommon  to  find  large 
trees,  especially  in  virgin  forests,  with  all  branches  but  one,  in  the  usual 
or  normal  position,  the  exceptional  branch  having  assumed  the  vertical 


2l6  PHARMACEUTICAL  BACTERIOLOGY 

trunk  position  and  being  tree-like  in  every  respect,  excepting  that  it  is 
devoid  of  a  root  system.  In  some  examples  of  this  kind,  the  lower 
branches  assume  the  root  position,  that  is  they  extend  downward  and 
the  secondary  branches  assume  the  more  irregular  positions  of  root 
branches,  as  compared  with  normal  secondary  branches  of  the  tree. 

It  may,  however,  be  that  many  of  these  growth  phenomena  are  not 
directed  and  controlled  by  enzymes  but  by  other  substances,  for  example, 
the  hormones.  Hormones  are  distinguished  from  enzymes  by  not  being 
destroyed  by  heat,  up  to  the  boiling  point;  they  are  dialyzable  and  in  a 
general  way  manifest  the  characters  of  chemicals.  They  occur  in  the  duct- 
less glands,  in  the  ovaries,  testes  and  in  other  organs.  It  is  known  that 
the  removal  of  the  organs  named  causes  very  serious  disturbances  in  the 
bodily  functions.  The  adrenal  and  pituitary  glands  are  apparently 
absolutely  essential  to  life  as  their  removal  in  animals  results  in  death. 
The  removal  of  the  pancreas  results  in  diabetes  and  the  removal  of  the 
ovaries  and  testes  produces  a  very  marked  change  in  general  metabolism 
as  well  as  in  the  mental,  muscular  and  nervous  systems,  with  arrest 
in  sexual  development.  Some  of  the  bodily  secretions  containing  these 
hormones1  are  now  being  used  in  the  treatment  of  certain  pathological 
conditions,  with  very  gratifying  results.  It  must  however,  be  admitted 
that  our  knowledge  of  the  exact  composition  and  function  of  hormones  is 
as  yet  not  well  understood,  nor  do  we  know  their  true  relationship  to  the 
enzymes. 

A  ferment  or  enzyme  may  be  defined  as  a  peculiar  energizing  substance, 
formed  by  living  cells  with  which  it  is  more  or  less  intimately  combined  or 
associated  but  without  being  vitally  influenced  in  its  activities  by  the 
vital  processes  of  the  cells  producing  it.  This  energizing  substance  or 
ferment  has  the  power  of  converting  the  latent  (potential)  energies  of 
chemical  compounds  into  kinetic  energies,  as  warmth  and  light.  The  new 
compound  or  compounds  formed  always  have  a  lower  kinetic  energy 
or  oxidizing  power  than  the  original  substance.  The  ferment  itself 
remains  unchanged  during  the  process.  Ferments  are  specific  in  their 
action,  that  is,  each  ferment  acts  upon  certain  substances  only  and  its 
activities  give  rise  to  constant  decomposition  products. 

Certain  ferments  (hydrolytic)  have  the  power  of  taking  up  moisture 
and  again  giving  it  up  to  the  substance  undergoing  fermentation,  the 
presence  of  moisture  being  necessary  to  the  process.  But  why  the  fer- 

1  Among  the  more  important  remedial  agents  of  this  group  are  extracts  of  the  para- 
thyroids, the  testes,  the  pituitary  bodies,  the  thymus,  the  ovaries,  the  mammary  glands, 
the  adrenals  and  the  pineal  gland,  all  of  which  are  now  marketed  and  used  with  con- 
siderable success  in  more  or  less  specific  ailments.  These  will  be  considered  in  another 
chapter. 


ZYMOLOGY — FERMENTS  AND  FERMENTATIONS        2i*J 

ment  takes  up  the  moisture  and  again  gives  it  up  is  as  yet  not  explained. 
Ferments  are  variously  influenced  in  their  activities  by  physical  and  chem- 
ical agents.  It  is  known  that  certain  chemicals  which  are  not  normally 
present  in  living  cells  or  organisms  and  which  are  not  component  parts 
thereof,  appear  to  have  a  stimulating  effect  upon  the  life  processes  of  these 
cells  or  organisms.  For  example  the  spores  of  Aspergillus  repens  will  not 
germinate  in  pure  water  or  in  inorganic  nutrient  solutions,  or  even  in 
peptone  solutions,  unless  some  inorganic  salt,  such  as  saltpeter,  is  added. 
Recent  tests  made  show  that  radium  exerts  a  very  marked  influence  upon 
plant  growth  as  well  as  upon  the  functional  activities  of  animal  cells  and 
organs. 

Minute  doses  of  certain  toxic  agents  have  a  stimulating  effect  upon  the 
vital  processes  of  lower  as  well  as  higher  organisms.  1-500,000  parts  of 
mercuric  chloride,  or  the  merest  trace  of  iodine,  of  potassium  iodide,  of 
chromic  acid  or  of  salicylic  acid,  have  a  very  beneficial  effect  upon  pro- 
cesses of  fermentation.  In  a  general  way  ferments  are  less  susceptible 
to  the  influence  of  poisonous  agents  than  are  the  microbes  or  other  organ- 
isms which  form  the  enzymes.  It  is  possible  through  the  judicious  use  of 
certain  antiseptics  to  kill  bacteria  and  other  objectionable  organisms  with- 
out in  any  way  hindering  the  fermentation  induced  by  associated  organ- 
isms. This  discovery  proved  of  great  value  in  experimenting  with  and 
isolating  enzymes.  The  following  substances  may  be  used  for  this  pur- 
pose— alcohol,  ether,  etheral  oils,  dilute  acids,  salicylic  acid,  thymol, 
chloroform,  calomel,  corrosive  sublimate,  etc.  Many  of  these  germ 
destroying  agents  are  however  not  without  some  checking  influence  upon 
the  action  of  the  ferments  themselves,  notably  salicylic  acid,  phenol, 
thymol  and  chloroform.  Toluol  appears  to  have  the  least  injurious 
effect  upon  enzymes. 

Ferments  influence  each  other.  Pepsin  inhibits  the  action  of  nearly 
all  other  ferments,  notably  that  of  trypsin  and  of  diastase.  Trypsin 
again  destroys  zymase.  Pepsin  has  however  only  a  slight  check  upon 
lactic  acid  fermentation.  Weak  acids  and  occasionally  also  weak  alkalies, 
have  the  power  of  converting  the  inactive  proferments  into  active  fer- 
ments, which  substances  Oppenheimer  designates  as  zymoplastic.  Other 
agents  as  warmth,  dilute  acids  and  alkalies,  increase  the  activity  of  fer- 
ments. Still  other  agencies,  as  cold,  most  alkalies,  alcohol,  etc.,  inhibit 
their  action  while  the  socalled  zymolytic  agents  (strong  acids,  heat,  etc.) 
kill  them.  Other  more  specific  properties  of  ferments  will  be  given  under 
the  description  of  the  ferments  themselves.  The  following  classifica- 
tion of  the  more  common  ferments  will  make  clear  their  relationship 
and  will  also  serve  as  an  introduction  to  the  specific  descriptions  which 
follow: 


2l8  PHARMACEUTICAL  BACTERIOLOGY 

CLASSIFICATION  OF  FERMENTS 
A.  HYDROLYTIC  FERMENTS 
I.  Proteid  Ferments. 

1 .  Proteolytic  ferments  (proteases) 

a.  Pepsin, 

b.  Trypsin 

c.  Lysins  (?) 
Bacterolysin, 
Hemolysin 
Cytolysin 

d.  Opsonins  (?) 

e.  Papain   - 

f.  Of  ductless  glands  (hormones)  (?) 

g.  Of  insectivorous  plants 

h.  Of  cryptogamous  plants  (bacteria,  higher  fungi,  etc.) 
i.  Seed  ferments 

2.  Coagulating  Ferments 

a.  Rennet 

b.  Vegetable  rennet 

c.  Agglutinins 

d.  Precipitins 

e.  Fibrin  ferments 

f.  Pectase 

3.  Starch  Splitting  Ferments  r 

a.  Diastases  (amylases) 
Amylase 

Ptyalin 

Amylopsin 

Glycogenic  ferment  of  liver,  blood  and  urine 

b.  Disaccharide  (diastase)  ferments 

Maltase 

of  yeast 

of  other  cryptogams 

of  animals 
Invertase 

of  yeast 

of  other  cryptogams 

of  animals 
Trehalase 
Melicitase 
Melibiase 
Lactase 


ZYMOLOGY — FERMENTS   AND   FERMENTATIONS  2IQ 

c.  Polysaccharide  (diastase)  ferments 
Cellulase  or  cytase 
Inulase     . 
Pectinase 
Seminase 
Cerubinase 
4.  Glucoside  Splitting  Ferments  (Glucases) 

a.  Emulsin 

b.  Myrosin 

c.  Gaultherase 

d.  Rhamnase 

5.  Fat  Splitting  Ferments — Lipases  (Steapsin) 

6.  Ammonia  Forming  Ferments — Urease 

7.  Lactic  Acid  Forming  Ferments 

B.  THE  OXIDIZING  FERMENTS 

1.  Zymases 

2.  Oxydases 

3.  Acid  Forming  Ferments 

a.  Vinegar  (acetic  acid) 

b.  Oxalic  acid 

c.  Citric  acid 

d.  Malic  acid,  etc. 

A.  Hydrolytic  Ferments 

The  socalled  hydrolytic  ferments  cause  the  splitting  of  complex  mole- 
cules accompanied  by  the  taking  up  of  H2O.  The  oxidation  processes  are 
of  an  intramolecular  nature,  that  is,  no  oxygen  is  taken  up  from  the  out- 
side (air). 

I.  Proteolytic  Ferments  (Proteases). — These  ferments  have  the  power 
of  splitting  albuminoid  substances.  Nothing  definite  is  known  regarding 
the  chemical  processes  involved  for  the  reason  that  but  little  is  known 
regarding  the  chemical  composition  of  albuminous  substances.  We  must 
rest  content  with  a  partial  study  of  the  end  products  of  the  fermentative 
processes.  The  following  are  the  more  important  ferments  of  this  group : 

i.  Pepsin. — Pepsin  is  the  albumen  digesting  ferment  found  in  the 
stomach  of  vertebrate  animals,  though  it  appears  to  be  wanting  in  some 
fishes.  Pepsin-like  ferments  are  however  also  found  in  insects  and  other 
invertebrates.  Pepsin  occurs  furthermore  in  the  intestinal  tract,  in 
muscle,  in  skin  secretions,  probably  in  other  tissues  and  organs,  and  in 
urine.  However  the  pepsins  of  difterent  species  of  animals  differ  some- 
what. It  is  also  noteworthy  that  the  pepsin  secretion  of  one  and  the 


22O  PHARMACEUTICAL  BACTERIOLOGY 

same  animal  is  most  variable,  depending  upon  the  nature  of  the  food 
ingested,  the  stage  of  the  digesting  process,  the  acidity  of  the  stomach, 
abnormal  or  pathological  conditions  as  in  gastritis,  cancer,  etc. 

The  "ingluvin"  of  the  older  pharmacopoeias  and  materia  medicas, 
is  the  pepsin  ferment  obtained  from  the  gizzard  of  the  domestic  fowl 
which,  at  one  time,  enjoyed  an  extensive  use  in  medicine.  The  pepsin  of 
the  dog  is  said  to  be  the  most  active.  That  of  the  frog  and  of  cold-blooded 
animals  generally,  is  less  sensitive  to  cold.  For  example,  frog  pepsin  is 
still  active  at  o°  C.,  while  the  pepsin  of  warm-blooded  animals  is  inac- 
tivated at  10°  C. 

Pepsin  is  not  immediately  elaborated  in  the  so-called  peptic  cells  of 
the  stomach,  rather  these  cells  form  a  proferment  or  pepsinogen  which 
in  association  with  dilute  free  hydrochloric  acid,  is  quickly  converted  into 
pepsin. 

Pepsin  has  never  been  isolated  in  a  pure  state.  In  its  comparatively 
purest  state  thus  far  obtained  it  is  a  yellowish,  brittle,  homogeneous  mass, 
soluble  in  water  and  dilute  solutions  of  acids,  solutions  of  salts  and  in 
glycerin.  It  is  precipitated  by  alcohol  and  has  the  general  properties 
of  enzymes.  It  is  quickly  inactivated  by  alkalies.  In  solutions  it  is 
destroyed  at  a  temperature  of  55°  to  57°  C.,  while  in  the  dry  state  it 
can  withstand  a  temperature  of  100°  C. 

Since  the  stomach  cells  contain  pepsinogen  very  largely,  the  washed 
stomach  pepsin  extract,  as  formerly  prepared  is  comparatively  inactive. 
To  determine  the  activity  or  digesting  power  of  pepsin  it  is  permitted 
to  act  upon  albuminous  substances.  Small  amounts  of  pepsin  dissolved 
in  dilute  solutions  of  hydrochloric  acid  (o.io  to  0.20  per  cent.)  are  allowed 
to  act  on  egg  albumen  or  fibrin.  The  time  required  for  a  unit  amount  of 
the  pepsin  solution  to  digest  a  unit  amount  of  the  albumen  at  a  given 
temperature,  uniform  for  all  tests,  indicates  the  activity  of  the  pepsin. 
Griinhagen  permits  a  small  mass  of  fibrin  to  become  saturated  with  the 
acidulated  pepsin  solution  and  then  places  the  mass  upon  a  filter.  The  num- 
ber of  drops  of  digested  fibrin  which  pass  through  the  filter  in  a  given  time 
indicates  the  digesting  power  of  the  pepsin.  Mett  places  small  glass 
tubes  filled  with  coagulated  albumen  into  the  pepsin  solution  in  an  incubator 
and  notes  the  amount  of  albumen  digested  in  ten  hours. 

The  action  of  pepsin  is  retarded  by  chloroform,  strong  solutions  of  salts 
generally,  particularly  sodium  chloride,  and  by  ammonium  sulphate. 
Alcohol  below  10  per  cent,  does  not  interfere  with  its  action,  while  beer 
even  with  only  3  per  cent,  alcohol  retards  its  activity  very  much.  Other 
substances  which  retard  pepsin  digestion  are  wine,  saccharin,  tea  and  coffee 
(due  to  tannin  present),  tobacco  and  strong  solutions  of  alcohol.  Weak 
solutions  of  acids,  small  amounts  of  spices,  very  minute  doses  of  arsenic, 


ZYMOLOGY — FERMENTS   AND   FERMENTATIONS  221 

strychnin  and  alkaloids  generally,  and  quinin,  assist  the  action  of  pepsin. 
Antipyrin  and  antifebrin  and  other  similar  coal  tar  derivatives  retard  its 
activity  somewhat  while  strong  solutions  of  cane  sugar  (40  per  cent.)  check 
its  activity  considerably.  Some  authorities  ascribe  a  bacteriolytic  power 
to  pepsin,  whereas  others  declare  that  the  apparent  germ-destroying  power 
is  due  to  the  acid  present. 

The  first  change  in  pepsin  digestion  is  a  swelling  of  the  albumens  or 
albuminoids,  which  action  is  due  to  the  acid  present.  The  pepsin  then 
splits  up  the  albumens  into  peptones  and  albumoses  which  are  diffusible. 
The  peptones  are  the  true  end  products  and  differ  from  the  albumoses 
in  that  they  are  more  diffusible  and  that  they  are  not  precipitated  by  acid- 
ulated ammonium  sulphate,  neither  are  they  precipitated  by  boiling, 
by  acid  s  or  by  calcium  ferrocyanide.  Pepsin  digestion  is  indeed  a  complex 
process  and  the  student  desiring  further  information  is  referred  to  standard 
works  on  physiology  and  dietetics. 

Commercial  pepsin  is  obtained  from  the  stomachs  of  recently  killed 
hogs.  Its  digesting  power  is  based  upon  its  proteolytic  action  upon  hard 
boiled  egg  albumen.  According  to  the  U.S.P.,  one  part  of  properly  pre- 
pared hog  pepsin  should  digest  at  least  3000  parts  of  coagulated  egg  al- 
bumen. Higher  grades  are,  however,  found  on  the  market,  such  as  give 
1-4000,  1-5000  and  1-6000  and  even  1-20,000. 

2.  Trypsin. — Trypsin  is  the  albumen  digesting  ferment  of  the  pan- 
creas derived  from  a  proferent,  the  trypsinogen.  It  is  also  found  in  the 
small  intestine.  On  its  action  on  albuminous  substances  it  is  much  like 
pepsin,  though  in  its  behavior  with  certain  modifying  influences  it  is  quite 
different.  It  is  most  active  in  slightly  alkaline  media  (one  per  cent,  solu- 
tion) though  it  is  also  active  in  neutral  and  slightly  acid  solutions.  Bile 
aids  trypsin  digestion,  especially  in  the  presence  of  lactic  acid,  also  in 
the  presence  of  hydrochloric  acid.  Strongly  acid  and  alkaline  solutions 
check  its  action  very  promptly  while  neutral  salt  solutions  increase  its 
activity,  especially  the  sodium  salts. 

Trypsin  is  somewhat  more  resistant  than  pepsin,  in  most  of  its  general 
characteristics  and  properties  it  is,  however,  closely  similar.  Its  digesting 
power  can  be  determined  much  in  the  same  manner  as  for  pepsin.  Trypsin 
is  especially  active  in  the  digestion  of  casein  and  of  gelatin. 

The  commercial  article  pancreatin  is  a  mixture  of  the  enzymes  of  the 
pancreas,  of  the  hog,  the  ox,  and  of  other  animals.  Its  chief  value  depends 
upon  its  peptonizing  and  diastatic  power.  According  to  the  French 
Pharmacopoeia  pancreatin  shall  peptonize  50  times  its  weight  of  fibrin 
and  convert  40  times  its  weight  of  potato  starch  into  sugar.  The  active 
ferments  of  pancreatin  are  trypsin  (protease),  amylopsin  (amylase), 
steapsin  (lipase)  and  the  milk  curdling  agent  rennin. 


222  PHARMACEUTICAL  BACTERIOLOGY 

3.  Lysins. — These    will  be  discussed  under  immunity  from  disease. 
Though   these   cell,  germ  and  blood  corpuscle  destroying  agents  found 
in  the  serum  of  the  blood  and  in  tissue  cells,  are  not  generally  classed 
as  ferments,  they  have  many  of  the  characteristics  of  the  proteolytic  fer- 
ments pepsin  and  trypsin.     Three  kinds  of  blood  lysins  have  been  studied, 
bacterolysins  which  actively  destroy  bacterial  cells,  hemolysins  which 
actively  destroy  red  blood  corpuscles  and  cytolysins  which  actively  de- 
stroy tissue  cells.     They  are  all  specific  in  nature  as  will  be  explained 
elsewhere. 

4.  Opsonins. — These  agents  found  in  the  blood  and  in    tissue  cells 
will  be  more  fully  described  elsewhere.    Like  the  lysins  they  are  specific 
in  action  and  their  enzyme-like  nature  is  rather  problematical. 

5.  Papain. — This  is  a  proteolytic  ferment  found  in  the  fruit  of  Carica 
papaya,  having  a  very  marked  action  on  meats.     It  was  used  by  the  natives 
of  Brazil  as  an  aid  in  preparing  meat.     Its  action  is  closely  similar  to  that 
of  pepsin.     Papain  is  a  well-known  commercial  product  used  in  defective 
stomach  digestion  and  also  for  the  purpose  of  peptonizing  meats.     It  does 
not  attack  living  protoplasm  hence  it  is  non-toxic.     A  papain-like  ferment 
is  found  in  the  fig,  in  the  pineapple  (bromelin),  in  cucumbers  and  in  other 
plants. 

6.  Of  Ductless  Glands . — The  ductless  glands  of  the  body  contain  certain 
substances  of  an  enzyme-like  nature  which  are  found  useful  in  the  treatment 
of  disease.     The  commercial  articles  made  from  these  glands  (adrenalin, 
desiccated  thyroid  glands,  etc.)  are  fully  described  in  standard  works  on 
materia  medica  and  in  dispensatpries,  to  which  the  reader  is  referred. 

7.  Of  Insectivorous  Plants. — Certain  plants  (Drosera  rotundifolia,  Nep- 
enthes   gracilis,  N.  hybridus,   Dioncea  muscipula,  Aldrovandia  vesiculosa, 
Utricularia  vulgaris   and   Darlingtonia  Calif  arnica)  secrete  a  pepsin-like 
ferment.     At  one  time  it  was  supposed  that  bacteria  symbiotically  as- 
sociated with  the  insectivorous  plant  hosts  secreted  the  proteolytic  en- 
zyme,  but  this   theory  has  recently  again  been  questioned.     There  is, 
however,  no  doubt  that  the. plants  named  have  the  power  of  digesting  and 
assimilating  animal  substances. 

8.  Of  Cryptogamous  Plants. — Proteolytic  ferments  are  found  in  some 
of  the  fungi  as  Penicillium,  in  Aspergillus  niger,  Agaricus  and  mFuligo 
septica.     Similar  ferments  are  found  in  many  different  species  of  bacteria 
as   anthrax   bacillus,    cholera    bacillus,    Bacillus   mesentericus   vulgatus, 
tubercle  bacillus,  sarcina  and  others.     Some  of  these  bacterial  ferments 
behave  like  exotoxins  in  that  they  are  absorbed  into  the  culture  media  in 
which  the  bacteria  are  grown.     Many  bacteria  have  the  power  of  liquefy- 
ing gelatine.     Some  of  the  yeasts  form  proteolytic  enzymes. 

9.  Seed  Ferments. — Proteolytic   ferments   are   widely   distributed   in 


ZYMOLOGY — FERMENTS   AND   FERMENTATIONS  223 

seeds,  their  function  being  to  split  up  and  render  transfusable  and  assimi- 
lable for  the  germinating  seeds,  the  proteid  granules.  These  have  thus 
far  not  received  any  careful  study. 

The  subject  of  auto-digestion  of  organs  has  received  considerable 
attention.  Finely  chopped  fresh  organs  digested  for  a  time  at  moderate 
warmth  undergo  certain  fermentation-like  changes  resulting  in  the  forma- 
tion of  reducing  sugars,  leucin,  tyrosin,  etc.,  substances  which  do  not  occur 
in  living  organs  or  in  organs  which  have  been  boiled.  Bacterial  infection 
is  excluded  by  means  of  chloroform  water  (using  ten  volumes  of  the  chloro- 
form water),  also  by  means  of  sodium  fluoride  and  thymol,  though  these 
latter  agents  are  less  suitable  than  the  chloroform  water. 

Autodigestion  proceeds  slowly,  being  a  slow  decomposition  of  the 
albuminous  matter.  In  this  digestion,  albumoses  are  formed  but  not 
peptones;  furthermore,  nuclein  is  split  up  which  is  not  the  case  in  trypsin 
fermentation.  Autodigestion  is  no  doubt  due  to  a  proteolytic  ferment 
which  probably  exists  in  the  cells  but  which  may  be  removed  from  the 
tissues  as  its  presence  has  been  demonstrated  in  cell-free  extracts.  The 
ferment  of  autodigestion  is  probably  an  autolytic  product  of  tissue  cells 
causing  a  molecular  change  in  albuminous  matter. 

2.  Coagulating  Ferments. — The  curdling  of  milk  was  known  in  ancient 
times  and  is  the  initial  basic  process  in  cheese-making.  At  one  time  it 
was  believed  that  pepsin  had  the  power  of  curdling  milk  but  that  is 
now  known  to  be  incorrect.  Berzelius  was  the  first  to  demonstrate  that 
the  curdling  of  milk  could  take  place  without  the  presence  of  lactic  acid, 
thus  disproving  the  contention  of  Liebig  that  lactic  acid  combined  with 
the  alkali  with  a  precipitation  of  casein. 

a.  Rennet. — Rennet  or  chymosin  is  the  milk-curdling  ferment  used  in 
cheese-making,  obtained  from  the  fourth  ventricle  of  the  stomach  of  the 
calf  or  sheep.  It,  however,  also  occurs  in  the  stomach  of  all  animals. 
In  serious  pathological  conditions  of  the  stomach  (as  cancer,  gastritis, 
etc.)  it  may  be  partially  or  even  wholly  wanting.  It  also  occurs  in  the 
intestinal  tract  and  in  nearly  all  tissues  and  organs. 

While  rennet  has  many  of  the  properties  of  ferments  generally,  it 
shows  some  exceptional  peculiarities.  In  regard  to  its  behavior  with  salt 
solutions,  it  is  precipitated  by  lead  acetate  only.  It  is  destroyed  by  bile 
and  by  even  very  weak  solutions  of  alkalies.  Dissolved  in  distilled  water 
it  is  destroyed  upon  exposure  to  a  temperature  of  40°  C.  This  peculiarity 
makes  it  possible  to  free  pepsin  from  rennet,  as  pepsin  is  not  affected  by 
this  temperature  and  distilled  water. 

Rennet  is  the  product  of  a  proferment  or  rennet  zymogen  which  is 
secreted  by  the  cells  of  the  stomach,  acted  upon  by  the  free  acid  of  the  stom- 
ach. Almost  any  acid  will,  however,  activate  the  zymogen,  especially 


224  PHARMACEUTICAL  BACTERIOLOGY 

hydrochloric  and  sulphuric  acid.  Acetic  acid  is  the  least  active.  Ren- 
net does  not  occur  in  the  stomach  in  the  absence  of  free  acid  though  the 
proferment  is  present. 

Rennet  is  a  highly  potent  ferment.  It  will  coagulate  from  4-800,000 
times  its  weight  of  milk.  It  is  most  active  in  slightly  acid  solutions  and 
least  active  in  alkaline  solutions.  Injecting  minute  doses  hypodermically 
causes  the  formation  of  an  anti-coagulating  ferment  which,  when  added  to 
milk,  prevents  coagulation. 

b.  Parachymosin. — The  rennet  of  the  human  stomach  and  that  of  the 
hog,  differ  from  that  of  other  animals,  especially  do  they  differ  from  that 
of  the  calf  and  sheep.     Thunberg  found  that  the  rennet  of  hog  pepsin 
does  not  coagulate  slightly  acid  milk  when  neutralized  by  alkali,  but  will 
do  so  when  such  milk  is  neutralized  by  means  of  carbonate  of  lime;  where- 
as calf  rennet  shows  no  such  difference  in  behavior.     It  is  therefore  pre- 
sumed that  in  addition  to  ordinary  chymosin,  the  pepsin  of  man  and  of  the 
hog  contains  a  modified  rennet  to  which  the  name  paracyhmosin  has  been 
given.     Parachymosin  is  more  resistant  to  heat  than  chymosin  as  it  is 
stilt  active  at  75°  C-,  while  it  is  much  more  susceptible  to  alkalies.     Heat" 
ing  hog  pepsin  to  70°  C.  destroys  the  chymosin  and  leaves  the  parachy- 
mosin  still  active,  or  the  parachymosin  may  be  destroyed  by  means  of 
alkalies  too  weak  to  affect  the  chymosin. 

c.  Vegetable  Rennet. — A  milk  curdling  ferment  occurs  in  various  plants. 
Galium  verum  is  used  to  curdle  milk,  also  Pinguicula  vulgaris,  Drosera  and 
Carica  papaya.     The  seeds  of  Wifharia  coagulans,  a  plant  found  in  India 
and  Africa,  are  used  by  the  Hindus  for  coagulating  milk,  their  religion 
forbidding  the  use  of  animal  substances.     Germinating  seeds  of  Ricinus 
communis  contain  rennet  in  the  form  of  the  zymogen  which  is  activated 
by  dilute  acids.     Rennet  also  occurs  in  figs,  artichokes,  thistle,  and  in 
other  plants.     The  fruit  of  Acanthosicyos  horrida,  of  South  Africa,  con- 
tains a  rennet  which  is  said  to  be  soluble  in  60  per  cent,  alcohol.     Nu- 
merous species  of  bacteria  form  milk  curdling  ferments,  among  others 
Bacillus    amylobacter,  B.    mesentericus  vulgatus,  B.    prodigiosus  and   B. 
cholera. 

d.  Pectase. — This  enzyme  causes  the  coagulation  of  pectin  in  plants 
containing  this  substance.     It  is  widely  distributed  among  higher  plants 
and  also  among  cryptogams.     It  occurs  in  two  forms,  soluble  as  in  carrots, 
and  in  an  insoluble  form,  as  in  acid  fruits  generally.     The  ferment  is  active 
in  the  absence  of  oxygen  and  it  does  not  result  in  gas  liberation,  and  is  most 
active  at  30°  C.     Coagulation  is  most  active  in  the  presence  of  lime, 
though  the  process  is  also  initiated  by  barium  and  strontium  salts.     Boil- 
ing or  precipitating  out  the  lime,  hinders  coagulation.     Acids  destroy 
the  ferment. 


ZYMOLOGY — FERMENTS   AND   FERMENTATIONS  225 

e.  Fibrin  ferment. — This  ferment  causes  the  coagulation  of  blood.     It 
occurs  in  the  blood  of  all  animals  excepting  in  "bleeders"  in  which  it  is 
absent,  a  condition  causing  much  trouble  in  checking  hemorrhages  result- 
ing from  even  very  trivial  injuries  and  operations.     It  is  supposed  that 
under  certain  conditions  the  fibrinogen  is  split  into  fibrin  and  globulin. 
The  fibrin  and  globulin  can  readily  be  separated,  as  is  done  in  the  manu- 
facture of  concentrated  diphtheria  antitoxin.     There  are  many  phases  of 
blood  coagulation  which  are  as  yet  not  understood.     Those  interested  will 
find  fuller  discussions  in  modern  works  on  human  physiology  and  anatomy. 

f.  Agglutinins    and  Precipitins. — These   problematical   enzymes   are 
found  in  the  blood  of  animals.     The  former  cause  the  bacterial  clumping 
as  in  the  Widal  test  for  typhoid  fever.     The  latter  cause  the  formation  of 
precipitates  in  blood.     Both  are  specific  in  nature  and  shall  be  more 
fully  discussed  elsewhere.     It  is  highly  questionable  whether  these  agents 
should  be  classed  as  coagulating  ferments. 

3.  Starch  Splitting  or  Sugar  Forming  Ferments. — Under  this  head  are 
included  those  highly  important  ferments  which  act  upon  carbohydrates, 
splitting  them  up  into  simpler  compounds.  The  fermentations  due  to 
these  enzymes  are  simple  hydrolytic  processes,  analogous  to  those  caused 
by  dilute  acids.  One  of  the  most  common  end  products  is  glucose. 

The  starch  splitting  ferments  are  widely  distributed  in  the  vegetable 
as  well  as  in  the  animal  kingdom  and  they  play  a  most  important  part  in 
domestic  economy.  They  bear  the  same  relationship  to  carbohydrates 
that  the  proteolytic  ferments  bear  to  proteid  substances. 

The  activity  of  starch  ferments  is  tested  in  various  ways.  Complete 
digestion  of  starch  is  indicated  by  the  absence  of  the  iodine  reaction.  The 
optical  behavior  of  the  substance  undergoing  fermentation  is  also  an 
indication  as  to  the  nature  and  identity  of  the  ferment.  Of  interest  is 
the  auxanographic  method  proposed  by  Beyerinck.  If,  for  example,  it  is 
desired  to  know  if  glucose  has  been  formed,  the  substances  is  inoculated 
with  a  fungus  which  will  develop  upon  glucose  (as  Saccharomyces  apicu- 
laius)  but  not  upon  maltose.  Fehling's  test  and  other  sugar  reactions 
may  also  be  used. 

The  saccharine  substances  formed  vary  in  composition  in  the  amount 
of  reduced  sugar  present,  in  the  character  and  degree  of  polarization 
(rotation),  etc. 

The  different  starches  as  of  rice,  barley,  wheat,  potato,  etc.,  do  not 
digest  or  ferment  at  the  same  rate  at  a  given  temperature.  For  example 
at  50°  C.  diastase  will  digest  12  per  cent,  barley  starch,  2  per  cent,  corn 
starch  and  29  per  cent,  malt  starch;  at  55°  C.,  5  per  cent,  potato  starch, 
53  per  cent,  barley  starch  and  58  per  cent,  malt  starch;  at  60°  C.,  52  per 
cent,  potato  starch,  92  per  cent,  barley  starch  and  18  per  cent,  corn  starch. 


15 


::: 


PHAXMACEUTICAL  BACTERIC 


At  very  low  temperatures  dbstasr  has  only  a  very  slight  effect  upon  raw 
potato  starch,  while  bailey  and  wheat  starches  are  quite  actively  digested. 
hese  are  the  true  starch  splitting  ferments  and  are 
or  amylorytk  ferments.  They  are  widely  distributed 
in  the  vegetable  and  also  in  the  animal  kingdom.  They  are  of  vital 

to  plants  and  inhnaK?  They  are  the  principal  tenner, 
the  so-called  malt  diastase  being  the  best  known  of  all  enzymes  or 
its.  The  discovery  of  malt  diastasr  dates  back  to  1853,  through 
the  investigations  of  Payen  and  Persoz,  who  sought  to  obtain  this  enzyme 
in  a  pure  state  by  precipitation  in  akohol,  but  in  this  attempt  they  were 
of  course  not  successful.  The  following  are  the  principal  disastatic 
Ferments. 

A  myLase. — This  is  «K««^»<^  proper  and  acts  on  starches,  rhanging  them 
It  is  very  widely  distributed  in  the  vegetable 
The  stored  starch  in  plants,  which  is  reserve  food,  must  be 
form  before  it  can  enter  into  circulation  through 
the  plant  iiancs  in  order  that  it  may  finaDy  be  assimilated  for  purposes 
of  plant  gum  lit  and  dtinrlmmmit  and  the  ferment  amylase  produces  this 
ability. 

— This  is  the  starch  frrmrnt  of  the  salivary  glands,  converting 
into  maltose.     It  is  a  very  active  ferment,  being  most  energetic 
um  and  is  quickly  destroyed  by  acids.     Some 
very  snail  quantities  of  hydrochloric  acid  in- 


the  action  of  ptyatin.     However,  0.015  per  cent,  acid  is  sufficient  to 


. — This  is  the  diastase  ferment  of  the  pancreas,  closely 
ibling  ptyalin.  though  it  is  more  active,  rapidly  liquefying  the  starch 
t  into  dfTtrhi  and  maffirir  It  is  most  active  between  30° 
and  45°  C.  and  is  destroyed  at  65°  C.  It  is  not  found  alone  but  in  associa- 
tion with  trypsim  g*»«p™i  and  icnniii  in  ri^  rnmnuTrLil  product  known  as 


The  action  of  amyiopsm  is  reduced  by  weak  acids,  but  on  the 

— Thegtycogenic  function  of  the  fiver  is  due  to  a  ferment 
starch  into  glycogen  and  dextrin-like  substances. 
found  in  the  blood,  in  urine  and  in  tissue  cells. 
(DUsUst)  Ferments. — These  ferments  act  upon  biose 

The  principal  enzymes 


ntdni 


. — This  is  very  widely  distributed  in  the  vegetable  kingdom, 
of  Saccharomyces  and  in  other  cryptogams.     It  also 
It  acts  upon  m*ha*+t  converting  it  into 


zno&OGT 


AND 


227 


Intertask. — Like  •»al<a«^  this 
table  and  aTvm«al  kingdoms.    It 

LI  -;  -".  -..--.  ~_-~~-     :  _'  _ : : -- 
•abase 


:: 


the 

or  tigala; ,  into  two 


This 
bottom  yeasts,  but  is  said  to  be 

into  d-gaiactose  a«H  d-g^acose,  in 


[frttf* — T-gfta<^  acts  on 

iifdi  :i_ir: :.   LT.~:    :-r__;.-c 


— 


no  lactase  but  it  occurb  in  the 

of  fully  grown 
is  also  said  to  occur  in 
scarcity  of 
thrdy  kss  fiable  to 

<L  Polsmcchmde  (Dimsttse) 


related  to  the 
are 

5:::_r-:     :  1.2.7.- 


off  many  of  oor 

ot  acted 
and  trehahse,  with  the 

of  bitter 

.—This  is  the 
It  is  widely 

iU'-e:  y'A-:^  .^      r^    A^ 


PHARMACEUTICAL  BACTERIOLOGY 

it  occurs  principally  in  bitter  almonds,  in  the  leaves  of  Laurocerasus  and  in 
the  seeds  of  the  Rosaceae.  Among  the  cryptogams  emulsin  is  found  in 
AspergiUus  nigtr,  Pcnirittum  glaucum  and  in  many  other  species  of  higher 
fungi  and  also  among  the  bacteria. 

Emulsin,  also  known  as  synaptase,  splits  amygdalin  into  grape  sugar, 
benzaldehyd  and  hydrocyanic  acid,  in  the  presence  of  water.  It  is  most 
active  at  45°-5o°  C.,  and  is  destroyed  at  70°  C.,  although  hi  the  dry  state 
it  can  resist  a  temperature  of  100°  C.  for  several  hours.  It  is  destroyed  by 
alkalies  whereas  acids  merely  inactivate  it.  It  also  decomposes  the 
glucosides  arbutin,  salicin,  helicin,  gentiopicrin,  syringin,  phyllirin,  cyclamin 
apiin  and  convallarin.  It  does  not  act  on  populin,  solanin,  hesperidin, 
convallaramin,  convolvulin,  digitalin,  hederin  and  quercitrin.  Emulsin 
is  said  to  decompose  lactose  into  glucose  and  galactose,  thus  resembling 
lactase  in  its  action. 

b.  Myrosin. — This  is  the  ferment  active  in  mustard  and  also  in  other 
cruciferous  plants.     It  acts  (in  the  presence  of  water)  upon  the  glucoside 
of  mustard,  sinalbin,  converting  it  into  mustard  oil,  dextrose  and  sinapin 
sulphate. 

c.  Gaulthtrase. — This  enzyme  was  first  discovered  in  Gaultheria  and 
named  gaultherase;   but  it  is  also  present  in  Betula  species,  Spircea 
idmaria,  S.  Jilipendula,  in  Monotropa  hypopuys  and  in  other  plants.     It 
acts  only  upon  the  glucoside  gaultherin  (abundant  in  Gaultheria  pro- 
cumbens).    It  does  not  act  on  salicin  or  amygdalin,  thus  differing  from 
emulsin. 

d.  Rhamnase. — A  ferment  which  is  said  to  occur  in  the  seeds  of 
Kkamnus  infectoria  and  which  acts  upon  the  glucoside  xanthorhamnin, 
decomposing  it  into  rhamnin  (rhamnetin)  and  glucose.     Boiling  destroys 
the  ferment  converting  it  into  rhamniose   (a  trisaccharid)   and  other 
products. 

Indigo  formation  is  supposed  to  be  due  to  the  action  of  a  glucoside 
splitting  ferment  but  this  has  not  yet  been  proven.  It  is  known  that 
Indican  (the  glucoside  of  Indigof era  and  Isatis  species)  is  decomposed  into 
indiglucin  and  indigwhite  in  the  presence  of  chloroform  water,  whereas 
boiling  destroys  such  action,  this  tending  to  prove  that  the  ferment  is  not 
of  bacterial  origin  as  was  once  supposed. 

Other  glucoside  splitting  ferments  probably  exist  in  plants,  as  for 
example  in  the  cucumber.  Ecbattium  elaterium  contains  a  ferment  which 
presumably  splits  up  the  glucoside  elaterin  and  which  may  be  called 
eleterase. 

5.  Fat  Splitting  Ferments. — As  is  known  fats  are  esters  of  glycerin. 
Under  the  influence  of  the  fat  splitting  ferments  the  fats  are  decomposed 
into  free  fatty  acids,  and  glycerin.  The  fat  enzymes  have  been  long 


ZYMOLOGY — FERMENTS  AND   FERMENTATIONS  22Q 

known  and  are  called  Upases,  steapsins  or  lipolytic  ferments.  The  best 
known  is  the  steapsin  of  the  pancreas.  It  is  very  sensitive  to  the  action 
of  acids  and  also  to  sodium  chloride.  Steapsin,  like  that  of  the  pancreas, 
is  also  found  in  the  blood  and  in  the  liver  of  various  mammalian  animals, 
even  in  fishes  and  in  insects. 

Lipase  is  also  found  in  the  vegetable  kingdom,  as  for  example,  in  the 
seeds  of  Ricinus  communis  and  among  the  cryptogams  as  Aspergittus 
niger  and  Bacillus  fluerescens.  It  causes  the  decomposition  of  fats  in 
germinating  seeds  and  in  other  fat  bearing  plant  tissues  and  organs. 

6.  Ammonia   Forming    Ferments — Urease. — These   ferments   act   on 
exposed  urine  changing  it  from  an  acid  to  an  alkaline  body,  due  to  the 
decomposition  of  urea  into  ammonium  carbonate.      The  organisms  that 
form  urease  are  not  normally  present  in  urine.    They  are  widely  distrib- 
uted in  the  atmosphere,  producing  their  characteristic  changes  in  exposed 
urine.    Most  of  the  urease  forming  organisms  belong  to  the  bacteria,  as 
Micrococcus  urinte  and  Bacillus  urea.    The  latter  is  highly  thermophflous, 
begin  capable  of  resisting  a  temperature  of  90°  C.    Many  other  urine 
organisms  are  reported,  including  cocci,  bacilli  and  sardnx.    They  are 
all  aerobic  and  require  grape  sugar,  urea,  phosphorus,  sulphur,  potassium 
and  magnesium  for  their  growth. 

Certain  bacteria  are  very  active  ammonia  formers.  A  bacillus  which 
developed  on  shrimp  and  also  on  fish  formed  a  sufficient  amount  of  am- 
monia to  suggest  the  possibility  of  utilizing  this  organism  in  the  com- 
mercial manufacture  of  this  gas. 

7.  Lactic  Acid  Forming  Enzymes. — Lactic  acid  is  widely  distributed 
and  is  most  important  from  the  commercial  viewpoint.     It  is  the  result 
of  the  decomposition  of  a  variety  of  sugars,  induced  by  a  great  variety 
of  microorganisms  belonging  to  the  groups  bacilli,  cocci,  vibriones,  and 
sarcinae.    All  agents  which  inactivate  or  kill  the  organisms  named  also 
inactivate  or  destroy  the  lactic  acid  enzymes  produced  by  them,  as  cold, 
heat  (above  60°  C.),  disinfectants,  acids,  etc. 

In  spite  of  the  wide  distribution  of  lactic  acid  in  the  animal  and  vege- 
table kingdoms  and  the  fact  that  a  great  many  organisms  are  known  to 
form  or  liberate  lactic  acid,  no  lactic  acid  enzyme  has  as  yet  been  isolated 
or  induced  to  act  independent  of  living  organisms.  This  has  raised  the 
question,  does  a  lactic  acid  forming  enzyme  really  exist?  The  presumptive 
evidence  however  is  decidedly  in  favor  of  the  existence  of  such  a  ferment. 
It  is  known  for  example  that  some  of  the  lactic  acid  bacteria  when  grown 
on  a  sugar  free  medium,  for  a  long  time,  will  when  suddenly  transferred 
to  a  medium  containing  sugar,  not  have  the  power  of  forming  lactic  acid. 
However,  on  prolonged  culturing  in  sugar-containing  media,  this  lost 
power  is  gradually  regained.  This  behavior  may  be  explained  on  the 


230       .  PHARMACEUTICAL  BACTERIOLOGY 

supposition  of  an  interruption  in  the  development  (by  the  bacteria)  of 
the  lactic  acid  enzyme.  It  is  furthermore  demonstrated  that  very  minute 
doses  of  mercuric  chloride,  copper  sulphate  and  other  poisons,  will  cause 
an  increase  in  the  lactic  acid  forming  function  while  the  power  of  septa- 
tion  and  growth  of  the  lactic  acid  enzyme  organisms  is  very  materially 
decreased. 

B.  Oxidizing  Ferments 

The  oxidizing  ferments  also  known  as  oxidases  cause  the  oxidation  of 
various  organic  substances.  They  appear  to  act  as  carriers  or  trans- 
mitters of  oxygen  to  the  substances  undergoing  fermentation,  though  the 
exact  chemical  changes  involved  are  not  well  understood.  To  this  group 
probably  belong  a  great  variety  of  ferments  and  fermentation  processes, 
widely  distributed  in  the  plant  as  well  as  animal  kingdoms.  Laccase  is 
a  ferment  concerned  in  the  formation  of  a  lacquer  varnish  in  the  lac  tree 
of  Asia.  Tryosinase  found  in  certain  fungi  and  also  in  the  roots  of  certain 
higher  plants  has  the  power  of  oxidizing  tryosin  which  is  found  in  these 
plants. 

(Enoxidase  causes  the  wine  disease  known  as  "casse."  The  wine 
loses  its  red  color  with  the  formation  of  a  reddish-brown  precipitate. 
It  is  highly  probable  that  the  multitudinous  fermentative  changes  com- 
prised under  "ripening"  processes,  "sweating"  processes,  aroma  and 
flavor  formation  in  wines,  tobacco,  cheese,  etc.,  are  of  the  oxidizing 
variety,  besides  many  of  the  little  understood  fermentative  changes 
resulting  in  so-called  "diseases"  in  commercial  products  as  wines,  beers, 
tobacco,  cheese,  etc. 

C.  Alcohol  Forming  Ferments 

The  alcohol  forming  ferments  or  zymases  or  yeast  ferments  proper 
are  by  far  the  most  important  from  a  practical  commercial  standpoint. 
Zymases  act  upon  sugars  splitting  these  substances  into  alcohol  and  car- 
bonic acid  gas  thus  acting  upon  the  end  products  formed  by  the  diastasse. 

Zymases  are  formed  by  a  great  variety  of  plants  and  animals,  par- 
ticularly the  yeast  plants  (the  Saccharomyces)  and  Torula  species  and  their 
varieties.  The  enzymes  of  yeast  plants  may  be  isolated  or  separated 
from  the  living  cells  and  will  continue  their  fermentative  activities 
independently. 

Alcohol  fermentation  is  by  no  means  a  simple  process.  The  degree 
of  alcohol  production  and  of  by-product  formation  varies  greatly,  depend- 
ing upon  a  great  variety  of  factors  and  influences.  To  enter  into  a  fuller 
discussion  of  the  details  of  the  fermentative  processes  and  a  description 
of  the  organisms  involved  is  not  essential.  We  may  however  mention 
the  fact  that  the  number  of  sugar  bearing  substances  capable  of  under- 


ZYMOLOGY — FERMENTS   AXD   FERMEXTATIOSfS  231 

going  alcoholic  fermentation  is  legion, 
varieties   and    forms    of 
are  involved  in  the  alcoholic 

JcnOWSv      In 

sake  and  brandy  making,  etc.)  a, 
yeasts,  lower  yeasts,  wild  yeasts,  etc. 

ani 


Hie  following  axe  the  principal  yeast 
principal  fermentative  activity  of  *arii_    Hansen's 
the  genera  Saccharomyces  and  Torula  is  based  nj 
Sacrha  lomjccs   form  spores   (aa4  <m>oies 
rarely  eight),  whereas  Toruly  does  not.    This  is  perhaps  aot  a  pcartiraMc 


•  bn 


i  :-..:;,  ;o:r 


232  PHARMACEUTICAL  BACTERIOLOGY 

saturnus,  Klocker  (from  soil) 

acidi  lactici,  Grotenfelt  (a  curdling  yeast) 

fragilis,  Jorgensen  (in  Kephir) 

barkeri,  Saccardo  (in  Ginger  beer) 

ludwigii,  Hansen  (in  oak) 

comesii',  Covara  (millet  pedicles) 

octosporus,  Beyerinck  (on  dried  currants) 

mellacei,  Jorgensen  (top  yeast,  pleasant  odor; 

guttulatus,  Robin  (in  rabbit) 

capsularis,  Schionning  (in  soil) 

Torulas  occur  in  great  variety.  The  Levure  de  set  is  a  yeast  capable  of 
developing  in  10  to -15  per  cent,  sodium  chloride  solution. 

The  following  are  a  few  products  in  which  there  is  alcoholic  fermenta- 
tion, in  addition  also  lactic  acid  formation. 

Yoghurt. — This  is  a  Bulgarian  sour  thick  or  klabbered  sheep's  or  cow's 
milk.  The  milk,  as  in  the  other  similar  fermented  drinks,  is  first  boilt, 
afterwards  evaporated  to  one-half  its  volume,  then  cooled  to  about  45°C. 
and  the  ferment  (maya  or  podko  assa)  added.  The  maya  is  simply  the 
dry  milk  residue  from  a  previous  fermentation.  The  fermented  product 
has  a  sour  aromatic  taste.  The  most  important  organism  in  this  fermen- 
tation is  the  Bacillus  bulgaricus.  Other  bacilli,  cocci  and  yeasts  are 
also  present. 

Kephir. — Kephir  is  an  effervescent  alcoholic  sour  drink,  made  from 
cow's,  sheep's  or  goat's  milk,  by  the  Bulgarians  as  has  been  explained 
elsewhere.  Several  yeasts  or  torulae  and  bacteria  are  active  in  the 
fermentation  process.  Dispora  (Bacillus)  caucasica  and  several  species 
of  Streptococci,  associated  with,  the  Kephir  yeast,  are  the  principal  organ- 
isms found.  These  organisms  no  doubt  form  a  mutualistic  association 
resulting  in  the  formation  of  lactic  acid  and  alcohol  in  the  milk. 

Koumiss. — This  drink  is  similar  to  kephir,  made  from  mare's  milk, 
by  the  inhabitants  of  southern  Russia  and  of  Siberia.  The  active  organ- 
isms are  a  yeast,  a  lactic  acid  bacillus  and  another  species  of  bacterium 
which  appears  to  be  characteristic  of  Koumiss  and  which  appears  to  be 
active  only  in  the  association  with  the  other  organisms,  thus  also  indicat- 
ing a  mutualistic  association.  The  fermented  milk  contains  lactic  acid 
and  alcohol.  , 

Soya  Sauce. — This  Chinese  sauce  or  relish  is  made  from  the  fermented 
soya  bean  (Glycine  hispida).  The  beans  are  boiled  and  mixed  with 
parched  wheat  meal  and  acted  upon  by  the  fungus  Aspergillus  oryza. 
This  is  then  mixed  with  salt  and  water  and  allowed  to  ferment,  sometimes 
for  a  year.  The  product  assumes  a  rich  brown  color  and  a  characteristic, 
aroma.  It  is  then  put  in  bags  and  an  almost  clear  juice  is  expresseid 
which  is  then  further  clarified  and  pasteurized.  In  the  second  or  long 


ZYMOLOGY — FERMENTS    AND    FERMENTATIONS  233 

process  of  fermentation,  several  organisms  are  active,  along  with  A. 
oryza,  as  Saccharomyces  soya,  Bacillus  soya  and  Sarcina  hamayuchia. 

Mazun. — This,  like  kephir  and  koumiss,  is  fermented  milk,  usually 
of  the  cow  and  goat,  and  is  much  used  in  Armenia.  The  active  organisms 
are  a  yeast,  a  bacillus  apparently  identical  with  B.  subtilis  and  several 
lactic  acid  bacteria. 

Leban. — This  sour,  aromatic  drink,  is  closely  similar  to»mazun  and  is 
made  from  boilt  buffalo's,  cow's  and  goat's  milk,  in  Egypt.  It  is  said  to 
contain  less  alcohol  than  kephir.  Five  different  organisms,  evidently 
mutualistically  associated,  are  active  in  Leban  fermentation;  a  Strepto- 
coccus which  coagulates  milk  and  forms  lactic  acid  from  lactose,  another 
bacillus,  a  Diplococcus  which  ferments  glucose,  saccharose  and  maltose;  a 
streptococcus  which  hydrolyzes  lactose  and  another  yeast  organism 
which  can  ferment  glucose  and  maltose  but  not  lactose. 

Ginger  Beer. — This  is  a  fermented  sugar  solution  to  which  ginger  -is 
added.  The  essential  fermenting  organisms  are  a  Saccharomyces 
(S.  pyriformis)  and  Bacillus  vermiforme.  Mycoderma  aceti  is  also  present. 
The  two  essential  organisms  are  evidently  in  close  mutualistic  relation- 
ship. The  drink  resulting  from  the  fermentation  of  saccharine  solution 
is  acid  and  effervescing.  The  so-called  "ginger  beer  plant"  which  is 
simply  a  mass  or  matrix  of  the  active  organisms  is  used  to  start  a  new 
fermentation. 

6.  Cider  Making 

Acetic-acid  fermentation  in  wine  cider  and  other  fermented  alcoholic 
substances  is  initiated  by  the  Mycoderma  aceti,  collectively  known  as 
" mother  of  vinegar."  This  is  no  doubt  a  mixed  growth,  representing 
several  species  or  varieties  of  acetic-acid  forming  organisms.  While 
it  is  true  that  nature  invariably  inoculates  the  substances  named,  re- 
sulting in  the  production  of  vinegar,  it  is  customary  to  use  the  top  skin 
or  pellicle  (mother  of  vinegar)  on  vinegar  already  formed,  adding  it  to 
new  wine  or  cider  in  order  to  hasten  the  fermentation.  As  stated,  this 
is  not  a  pure  culture  representing  a  single  species.  In  fact,  the  tests 
with  what  were  pure  species  have  proven  unsatisfactory.  The  vinegar 
organisms  require  an  abundance  of  oyxgen.  To  supply  the  necessary 
oxygen  (of  the  air)  it  is  customary  to  have  the  fermentation  barrels  or 
casks  only  about  two-thirds  or  three-fourths  full  and  to  leave  the  bunghole 
open  (generally  with  a  plug  of  cotton).  In  Germany  a  quickened 
method  is  much  in  vogue.  The  wine  or  cider  is  allowed  to  trickle  slowly 
through  a  cask  filled  with  wood  shavings  which  are  moistened  with  old 
vinegar.  The  wood  shavings  offer  a  maximum  surface  exposure  and 
fermentation  is  as  a  result  very  much  hastened. 


234  PHARMACEUTICAL  BACTERIOLOGY 

Occasionally  the  vinegar  loses  its  acidity.  This  is  due  to  the  invasion 
of  a  bacillus  (B.  xylenum)  which,  in  the  presence  of  oxygen,  splits  up  the 
acetic  acid  into  other  compounds.  This  change  can  be  prevented  by 
excluding  air  from  the  containers.  Vinegar  should  contain  4  to  4.5 
per  cent,  of  acetic  acid  (the  legal  standard). 

Beer  and  Wine  Organisms.  —  We  have  elsewhere  briefly  outlined 
the  manufacture  of  beer  and  sak6.  Numerous  yeast  organisms  are  active 
in  wine,  cider  and  in  other  fruit  juice  fermentations.  The  student  desiring 
further  information  is  referred  to  the  works  by  Hansen  and  Jorgensen, 
which  may  be  found  in  any  library  of  scientific  books.  Cider  vinegar  and 
yeast  manufacture  have  been  mentioned  elsewhere,  likewise  cheese 
making  and  the  significance  of  dairying  organisms  in  the  ripening  of 
cream  and  of  butter,  etc. 

D.  Acid  Forming  Ferments 

As  is  known,  dilute  alcohol  upon  standing  exposed  to  air,  gradually 
becomes  sour,  losing  its  alcohol  more  and  more.  This  is  due  to  ferments 
which  act  upon  the  alcohol,  splitting  it  into  acetic  acid  and  H2O.  The 
organisms  producing  the  acid  forming  enzyme  are  generally  classed  with 
the  bacteria,  largely  in  the  group  Bacillus,  the  principal  species  being 
Mycoderma  (Bacittlis)  aceti,  B.  pasleurianum,  B.  kutzingianum,  B. 
oxydans,  and  B.  acetosum.  The  yeast  Saccharomyces  mycoderma  is  also 
capable  of  forming  acetic  acid.  The  vinegar  organisms  are  most  active 
at  25°  to  30°  C.  Very  slowly  active  at  10°  C.  and  killed  at  temperature 
but  slightly  above  35°  C.  The  so-called  mother  of  vinegar  consists  of 
an  agglutinated  mass  of  vinegar  organisms  and  is  used  as  a  starter  in  the 
manufacture  of  vinegar.  Thus  far  it  has  not  been  possible  to  isolate  the 
vinegar  ferment  as  has  been  done  with  diastase  and  zymase. 

There  are  acids  of  non-alcoholic  origin  formed  by  living  ferments,  such 
as  oxalic  acid,  malic  acid,  citric  acid  and  others,  which  appear  to  be  de- 
rived from  sugars  direct.  Citric  acid  is  formed  from  sugars  through  the 
activity  of  two  fungi,  Citromyces  pfejferianus  and  C.  glaber.  Saccharo- 
myces hansenil  forms  oxalic  acid  from  mannit  and  galactose,  without 
alcohol  formation. 

The  following  table  will  serve  to  make  clear  the  relationship  of  the 
diastase  (starch),  zymase  (sugar)  and  alcohol  ferments: 


Principal  foods  for  enzymes  detrins  Sugars  Alcohol 


Enzymes Diastases 


I 

Vinegar 

Zymases  ferments 

(yeasts)         (Mycoderma) 


Principal  Products Sugars  Alcohol          Acetic  Acid 


CHAPTER  X 
IMMUNOLOGY.    IMMUNITY  AND  IMMUNIZING  AGENTS 

i.  Introduction. — Immunity  from  disease  and  susceptibility  to  disease 
are  relative  terms.  By  immunity  is  meant  the  power  or  ability  of  the 
living  body  to  resist  or  prevent  the  successful  invasion  by  the  agent  or 
agents  of  infection,  whether  these  agents  are  of  vegetable  or  of  animal 
origin,  whether  they  are  the  organisms  themselves,  as  bacteria,  amebse, 
yeast  cells,  nematodes,  etc.,  or  the  substances  produced  by  them,  as 
toxins,  ptomaines,  albumins,  toxalbumins,  putrescins,  venoms  and 
enzymes.  The  body  resistance  to  the  action  of  chemicals  and  chemical 
poisons  is  generally  not  classed  as  immunity,  although  it  is  a  closely 
related  phenomenon  and  cannot  well  be  omitted  from  the  discussion. 

We  know  that  there  is  great  variation  in  the  immunity  of  the  indi- 
viduals of  the  same  species  to  the  various  agents  of  infection  as  well 
as  to  the  action  of  the  purely  chemical  poisons  of  non-living  or  inorganic 
origin.     A  number  of  individuals  exposed  to  the  same  infection  do  not 
all  take  the  disease.     A  number  of  individuals  receiving  the  same  dose 
of  poison  are  not  all  affected  in  the  same  degree  or  in  the  same  way.     It 
has  been  known  for  a  long  time  that  the  successful  invasion  of  certain 
infections  as  small-pox,  typhoid,  measles,  mumps,  whooping-cough,  etc., 
immunizes  the  individual  against  subsequent  attacks.     It  has  also  been 
known  for  ages  that  the  animal  organism  may  resist  gradually  increasing 
doses  of  highly  toxic  substances,  as  arsenic,  opium,  morphine,  tobacco, 
and  alcohol.     Even  more  remarkable  is  race  immunity.     Man  is  immune 
to  most  of  the  diseases  of  the  lower  animals,  as  hog  cholera,   chicken 
cholera,  and  on  the  other  hand  most  animals  cannot  be  successfully 
infected  by  the  human  diseases,  such  as  measles,  mumps,  whooping- 
cough,   scarlet    fever,   and  yellow  fever.     Closely  related    species  may 
display    remarkable    immunity    differences.     For    example,    field    mice 
are  very  susceptible  to  glanders,  whereas  the  common  house  mouse  is 
quite  immune.     Jersey  cows  are  less  liable  to  bovine  tuberculosis  than 
Holsteins.     The   Yorkshire   breed   of   swine   is   less    susceptible   to  hog 
erysipelas  than  are  other  breeds. 

235 


236  PHARMACEUTICAL   BACTERIOLOGY 

Trachoma,  measles,  poliomyelites,  typhus,  scarlet  fever,  rabies, 
influenza  and  whooping  cough,  can  be  transmitted  to  monkeys.  Small- 
pox can  be  transmitted  to  cows,  horses,  rabbits,  and  sheep.  Rabies  is 
transmissible  to  dogs,  wolves,  cows,  rabbits,  cats  and  other  animals; 
malta  fever  to  guinea  pigs,  mice  and  rabbits;  plague  to  most  domestic 
animals,  to  rats,  squirrels,  and  mice.  The  gonococcus  is  not  transferable 
to  lower  animals,  whereas  the  streptococcus,  the  staphylococcus  group  and 
the  pneumococcus  group  are  transferable  to  many  of  the  lower  animals. 
The  following  diseases  of  lower  animals  are  transmissible  to  man;  ring- 
worm, favus,  scabies,  tetanus,  anthrax,  glanders,  actinomycosis,  psit- 
tacosis (a  lung  disease  of  parrots),  plague,  trichnosis,  bovine  tuberculosis, 
foot  and  mouth  disease,  and  influenza.  Many  of  the  lower  animals 
harbor  the  primary  causes  of  diseases  of  man  without  suffering  any 
pronounced  or  even  appreciable  inconvenience.  Thus  the  oyster  harbors 
the  organisms  of  typhoid  fever  and  of  bacillary  dysentery.  Mosquitoes 
harbor  the  causes  of  malaria  and  of  yellow  fever. 

The  stable  fly  carries  the  primary  cause  of  poliomyelitis  and  the  rat 
flea  harbors  the  plague  bacillus.  The  differences  in  racial  reactions  to- 
wards poisons  is  also  remarkable.  The  hog  feeds  upon  poison  oak  and  is 
quite  immune  to  snake  venom  as  well  as  to  many  other  substances  which 
are  highly  toxic  to  man.  The  goat  is  immune  to  many  infections  and  feeds 
with  impunity  upon  many  poisonous  plants.  Herbivora  are  far  less 
susceptible  to  vegetable  alkaloids  than  are  carnivora.  Insects  feed  upon 
plants  which  are  highly  poisonous  to  man. 

The  Caucasian  is  more  susceptible  to  yellow  fever  than  is  the  negro, 
whereas  the  reverse  is  true  as  to  tuberculosis,  smallpox,  and  syphilis.  As 
compared  with  herbivora,  the  wild  carnivora  are  quite  immune  to 
tuberculosis  and  are  also  far  less  susceptible  to  other  infections.  Some 
infections  are  highly  humanized,  as  gonorrhea,  syphilis,  and  cancer. 

There  are  also  the  peculiarities  of  age  immunity,  sex  immunity,  clirna- 
tological,  occupational  and  seasonal  immunity,  etc.,  all  of  which  require 
the  attention  of  the  physician. 

Immunity  is  very  markedly  relative.  For  example,  no  race  of  man- 
kind is  possessed  of  absolute  immunity  to  any  human  disease.  The 
immunity  enjoyed  by  the  wild  carnivora  can  be  broken  down  by  pro- 
longed captivity,  notably  the  immunity  to  tuberculosis.  A  thousand  and 
one  factors  modify  immunity,  as  lack  of  food,  poor  food,  fatigue,  over- 
exertion,  cold,  excessive  heat,  dampness,  poisons,  habits,  occupations, 
etc.,  -etc. 


IMMUNOLOGY.      IMMUNITY  AND   IMMUNIZING   AGENTS 

The  several  kinds  of  immunity  may  be  tabulated  as  follows  : 


237 


Immunity 


Inherited 

(Natural) 


Racial 
(Phylogenetic) 


Individual 


As  observed  in  the  different  orders, 
families,  genera  and  species  of  the 
animal  kingdom. 

As  observed  in  different  individuals 
of  the  same  species  or  variety.  (On- 
togenetic.) 


As  observed  in  the  sexes  of  the  same 
species  or  variety. 


Induced 
(Acquired) 


Active 


Passivi 


Pathogenetic 
and  environ- 
mental 


Due  to  diseases  which 
produce  immunity  to 
subsequent  attacks 
as  diseases  of  child- 
hood; acclimatiza- 
tion; etc. 


Due  to  use  of  modified 
Artificial  J   toxins,  bacterins,  and 

direct  inoculation 
with  disease  germs. 
-Use  of  antitoxins  and  other  disease  preventives. 


2.  Earlier  Theories  Regarding  Immunity. — Theories  have  been  ad- 
vanced from  dm  to  time  which  were  intended  to  explain  immunity. 
Why  the  individual  and  racial  variation  in  the  behavior  towards  infections? 
Why  are  certain  individuals  successfully  infected  while  others  escape? 
Why  do  some  of  those  who  are  successfully  infected  die  while  others 
recover?  Why  do  the  infections  (in  the  cases  of  recovery)  disappear  after 
a  time?  The  following  are  som£  of  the  theories  advanced: 

a.  Physiological  Resistance  of  the  Body  Cells. — Some  thirty  to  forty 
years  ago,  physicians  and  physiologists  had  much  to  say  regarding  the 
variable  though  inherent  property  or  power  possessed  by  the  living  cells 
of  the  body  to  resist  or  ward  off  infection,  the  so-called  physiological  re- 
sis  tence  of  cells.     It  was  thought  that  as  long  as  the  cells  of  the  body 
were  fully  or  normally  active  the  infecting  agents  could  not  find  lodgment 
therein.     This  theoretical  assumption  cannot  be  gainsaid  even  at  the 
present  time,  but  unfortunately  the  theorization  says  nothing  and  ex- 
plains nothing.     The  theory  has  no  visible  means  of  support. 

b.  The  Exhaustion  Theory. — In  order  to  account  for  the  cessation  of 
successful  infections  in  cases  of  recovery,  it  was  assumed  that  the  infecting 
agents  (bacteria)  used  up  those  body -cell  substances  which  were  neces- 
sary to  the  life  of  the  particular  infection.     It  was  thought  that  these  cell 


::     ir.rr      : 


:I:-.T:-     -  r:i  "i  :_i".  ;    .:.-:..-.  :  ir. 


On 

the  experimental  cvi- 
that  as  a  result  both 
to  the  more  recent 


n .  r: :  rr   =_i  i 


:  -    ;    ~:     :  '  :  : 


or  destroyed  the 
To  this  substance  they  give  the 
It  led  to  the 

.•-.-;.:  ------- 


-  -  •.:-  -.>..:•- 


They  are  readily  destroyed 

(65*  to  75s  C)  and  by  ezoosiire  to 


to  acidi  ir.-;  >re  '.^t  preserved 

Kdtodi 
a  vacmmL,  at  a  low 


IMMUNOLOGY.      IMMUNITY  AND   IMMUNIZING  AGENTS  239 

sulphate  and  other  salts.  Remarkably  enough,  reactions  have  been  ob- 
served which  would  indicate  that  antitoxin  is  not  a  proteid  substance;  for 
example,  it  is  not  destroyed  (digested)  by  trypsin. 

It  has  furthermore  been  found  that  variably  gmall  amounts  of  antitoxin 
exist  in  normal  blood;  that  is,  in  the  blood  of  animate  that  have  not  been 
naturally  or  artificially  immunized,  and  also  in  still  lesser  amounts  in  the 
mi]  k  of  normal  animals .  As  to  the  origin  of  the  antitoxins  the  phvsk>1ogic 
evidence  points  to  their  formation  in  the  body  cells  rather  than  in  the 
blood  serum. 

Another  important  discovery  was  that  normal  blood  could  actively  de- 
stroy (lake)  bacteria,  and  in  common  with  antitoxins,  this  bactericidal 
property  was  found  to  be  specific.  That  is,  serum  found  to  be  quite 
destructive  to  the  typhoid  bacillus  is  not  destructive  to  the  cholera  bacil- 
These  germ  destroying  or  bactericidal  substances  are  designated 
lysins.  Ehrlich  has  discovered  that  there  are  in  fact  three  distinct  blood 
5;  namely,  cytolysin,  a  substance  which  is  capable  of  destroying 
(taking)  body  cells;  hemolysin,  which  is  capable  of  destroying  red  blood- 
corpuscles;  and  bacteriolysin  as  already  explained.  By  injecting  tissue 
cells,  as  those  of  kidney  or  of  some  other  organ,  into  an  animal,  there  are 
developed  in  the  blood  of  the  inoculated  animal  lysins  which  will  dissolve 
kidney  cells  or  other  organ  cells  used.  If  the  blood  of  a  bird  or  other 
animal  is  injected  into  an  animal  of  a  different  species,  hemorysms  will 
appear  in  the  blood  of  the  animal  thus  injected.  This  hemorysin  is  specific, 
as  it  wfll  only  dissolve  or  destroy  the  red  blood  cells  in  the  blood  of  the  kind 
of  animal  of  which  the  blood  was  used  for  injecting.  An  animal  inocu- 
lated with  the  typhoid  bacillus  will  produce  a  bacteriolysin  which  destroys 
the  typhoid  bacillus.  Lytic  sera  become  inactive  when  heated  to  55°  C. 
for  one-half  hour  and  such  sera  are  said  to  be  inactivated.  However,  if 
normal  serum  is  added  to  the  inactivated  serum  the  bactericidal  power  is 
fully  restored.  The  bactericidal  power  of  the  serum  can  be  greatly  in- 
creased by  the  use  of  highly  virulent  bacterial  cultures,  thus  producing  a 
serum  of  high  potency.  In  actual  practice,  as  in  the  manufacture  of 
bactericidal  sera  for  the  prevention  and  cure  of  disease,  the  animal  (as 
is  first  inoculated  with  attenuated  cultures,  then  with  normally 
virulent  cultures  and  finally  with  hyper-virulent  cultures  of  the  specific 
pathogenic  microbe.  Such  sera  act  by  destroying  the  disease-producing 
bacteria,  but  they  have  no  effect  upon  the  toxins  produced  by  the  bacteria, 
thus  showing  that  they  are  entirely  distinct  from  the  antitoxins. 

The  eminent  bacteriologist  Metchnikoff  made  the  very  interesting 
discoven-  that  the  white  blood-corpuscles  (leucocytes)  had  the  power  of 
feeding  upon  and  digesting  bacteria  with  which  they  came  in  contact. 
That  is  the  white  blood  corpuscles,  called  phagocytes,  act  as  the  defenders 


240  PHARMACEUTICAL  BACTERIOLOGY 

of  the  body  against  bacterial  invasion.  This  observation  by  Metchnikoff, 
fully  verified  by  others,  is  generally  known  as  the  phagocyte  theory  and  the 
phenomenon  is  designated  phagocytosis.  The  principle  involved  in 
phagocytic  activity  is  well  illustrated  in  the  lesser  local  injuries,  as  cuts, 
bruises,  abrasions,  etc.  Normally  such  injuries  are  always  infected  by 
various  germs  of  the  environment,  as  the  several  varieties  of  pus  microbes. 
These  invading  microbes  at  once  begin  their  attack  upon  the  tissue  cells 
and  blood-corpuscles.  The  leucocytes  which  are  present  begin  to  feed 
upon  the  rapidly  multiplying  pus  organisms  but  for  a  time,  as  a  rule,  the 
latter  have  the  upper  hand  and  as  a  result  there  is  perceptible  pus  forma- 
tion ("the  laudable  pus"  of  older  writers)  represented  by  dead  leucocytes 
gorged  with  microbes.  As  the  inflammatory  reaction  becomes  more 
marked,  indicated  by  redness  and  swelling  of  the  tissues  immediately  about 
the  injury;  increased  numbers  of  leucocytes  (phagocytes)  are  brought  to 
the  scene  of  action  and  gradually  they  gain  control  until  finally  the  invad- 
ing microbes  are  all  destroyed,  thus  permitting  a  rapid  and  unhindered 
restoring  of  tissue  cells,  recognized  as  the  healing  process.  This  phago- 
cytic action  is  entirely  distinct  from  the  action  of  antitoxins  and  lysins,  and 
the  three  are  potent  factors  in  immunity. 

The  investigations  of  Metchnikoff  and  Leishman  on  phagocytosis 
paved  the  way  for  the  discovery  of  opsonins  by  Wright.  It  was  noticed 
that  the  phagocytic  activity  was  influenced  by  conditions  to  be  found  out- 
side of  the  leucocytes  themselves.  Metchnikoff  held  that  the  principal 
part  is  played  by  substances  found  in  the  serum  and  in  the  tissue  cells  to 
which  he  gave  the  name  "  stimulins."  The  purpose  of  these  substances  in 
the  tissue  fluids  have  not  yet  been  satisfactorily  demonstrated,  but 
Metchnikoff  considers  their  function  to  be  that  of  acting  upon  the  phago- 
cytes in  such  a  manner  as  to  stimulate  them  to  perform  phagocytosis. 
Wright,  Hektoen,  Neufeld  and  others  have  demonstrated  beyond  doubt, 
the  presence  in  the  blood  of  substances  which  act  upon  the  infecting  bac- 
teria and  get  them  ready  for  the  completion  of  their  destruction  by  the 
phagocytes.  To  these  bodies  Wright  gave  the  name  " opsonins"  (Latin, 
opsono,  I  prepare  for).  That  opsonins  are  not  formed  in  the  blood  is  cer- 
tain. Experimental  evidence  seems  to  prove  that  they  are  products  of 
muscular  or  subcutaneous  cellular  activity.  It  is  probable  that  the  actual 
formation  of  opsonin  occurs  in  the  muscle  tissues  and  passes  thence  to  the 
blood.  Wright  has  demonstrated  more  or  less  satisfactorily  the  presence 
of  opsonins  in  the  blood  of  animals  and  humans  and  by  a  special  technic 
has  measured  the  relative  amount.  This  measurement  is  a  ratio  of  the 
activity  of  the  phagocytes  in  normal  blood  and  of  that  in  disease,  before 
and  after  stimulation,  determined  by  the  number  of  bacteria  that  a  single 
phagocyte  will  ingest — the  so-called  opsonic  index.  This  index  or  ratio  is 


IMMUNOLOGY.      IMMUNITY  AND   IMMUNIZING   AGENTS 


241 


made  intelligible  by  decimal  figures  representing  the  number  of  bacteria 
which  the  average  phagocyte  will  take  up.  We  may  assume  that  one 
phagocyte  in  normal  blood  will  ingest  an  average  of  10  bacteria,  repre- 
sented in  the  index  by  the  figures  i.o,  but  in  disease  (chronic)  the  phago- 
cytes may  only  take  up  an  average  of  3,  6,  or  other  numbers,  represented 
by  the  figures  0.3,  0.6,  etc.  After  stimulation  the  phagocytes  may  take 


FIG.  59. — Opsonic  Incubator.  The  determination  of  the  Opsonic  Index  has  become 
so  important  that  these  incubators  have  been  made  to  meet  the  demands  for  an  appa- 
ratus in  which  twenty  pipettes  can  be  incubated  at  one  time  and  so  that  any  tube  may 
be  examined  during  the  progress  of  the  experiment  without  changing  the  temperature  of 
the  others.  There  are  twenty  tubes  for'  opsonic  pipettes  and  an  extra  tube.  The  tubes 
may  be  easily  removed  when  dedred  by  means  of  a  key  which  accompanies  the  incubator. 
On  top  there  are  eight  tubes,  22  mm.  in  diameter,  for  test-tubes.  Each  is  provided  -with 
a  nickel-plated  cap.  The  incubator  is  supplied  with  thermometer,  thermo-regulator, 
and  a  two-flame  burner,  with  wire  guard. 

UP  I5)  25>  or  even  numbers  of  bacteria  represented  in  the  index  by  the 
figures  1.5,  2.5,  etc. 

Taking  the  opsonic  index  of  an  individual's  blood  cells  for  considerable 
delicate  technic.  In  brief,  it  is  performed  by  mixing  together  equal  vol- 
ume quantities  (measured  in  a  capillary  tube)  of  blood  serum  and  an  emul- 
sion of  bacteria  and  incubating  for  15  minutes  at  37.5°  C.  Then  making  a 
thin  smear  of  the  mixture  on  a  microscope  slide,  drying  and  staining,  and 
counting  the  number  of  bacteria  enclosed  in  each  white  blood-corpuscle 
(50  to  200  cells  counted)  and  striking  an  average.  This  average  is  the 

16 


242     -  PHARMACEUTICAL  BACTERIOLOGY 

index  stated  by  a  decimal  figure.  The  index  thus  obtained  indicates  the 
relative  phagocytic  power  of  the  individual's  blood  tested,  whether  below 
or  above  the  normal,  or  normal. 

The  opsonic  index  taken  in  the  various  chronic  forms  of  bacterial  infec- 
tions is  invariably  below  normal  and  shows  that  the  phagocytic  power  is 
low,  and  it  seems  to  prove  that  the  chronicity  is  due  to  the  subnormal 
phagocytosis.  The  injection  of  several  millions  of  devitalized  bacteria  of 
the  kind  causing  the  infection,  induces  .the  formation  of  the  specific 
opsonin,  arouses  the  phagocytic  activity  and  corrects  the  pathologic 
condition.  The  opsonic  method  of  treatment  has  been  extensively  tested 
through  the  use  of  specifically  active  bacterial  suspensions  (vaccines, 
bacterins  or  opsonogens)  which  in  some  instances  have  given  excellent 
results.  It  has  also  been  found  that  substances  other  than  opsonins  may 
increase  phagocytosis,  as  for  example,  nucleinic  acid  and  collargol. 

From  the  foregoing  it  becomes  evident  that  immunity  from  disease 
depends  upon  the  presence  in  the  body  of  antitoxins,  bacterolysins,  and  the 
opsonins  which  induce  phagocytosis.  It  is  furthermore  possible  to  increase 
the  activity  of  these  agents  artificially.  All  three  agents  are  specific  in 
nature  as  already  stated.  Ehrlich  has  attempted  to  explain  the  phenomena 
of  immunity  according  to  his  receptor  or  side  chain  theory  (Seitenketten- 
theorie).  This  theory,  which  is  rather  complex  and  highly  technical,  was 
first  used  to  explain  cell  metabolism.  Hinman's  version  of  the  side  chain 
theory  is  very  simple  and  we  give  it  as  follows:  As  applied  to  immunity 
the  basis  of  the  theory  is  the  conception  of  the  duplex  nature  of  antigens. 
An  Antigen  is  a  substance,  of  bacterial  or  other  origin,  which  has  the  £ower 
when  introduced  into  the  bodya  of  inducing  the  formation  of  specific  anti- 
bodies. Not  all  toxins  or  poisons  have  this  power.  For  example,  strych- 
nin and  the  toxin  of  tetanus  produce  similar  physiologic  effects,  but  only 
the  latter  is  capable  of  producing  an  antibody.  Ehrlich  explains  this 
difference  by  assuming  that  strychnin  and  most  other  vegetable  poisons 
enter  into  a  loose  combination  with  the  cell  plasm,  analogous  to  an  aniline 
dye  which  can  be  readily  dissolved  out  again;  whereas  the  toxin  is  firmly 
bound  to  the  cell,  representing  in  a  measure  a  toxic  food-stuff  in  chemical 
union  with  and  assimilated  by  the  cell.  The  atomic  combination  of 
the  toxin  antigen,  which  represents  this  chemical  union  is  designated 
the  haptophore  group,  while  the  atomic  combination  of  the  cell-plasm 
with  which  the  haptophore  group  unites  is  called  the  cell  receptor  group. 
The  haptophore  group  is  distinct  from  the  atomic  group  which  produces 
the  toxic  or  pathologic  effects,  designated  as  the  toxophore  group.  These 
two  groups  of  the  antigen  (toxin),  namely  the  haptophore  group  and 
the  toxophore  group,  act  independently  of  each  other  and  possess  different 
properties.  The  toxophore  group  is  easily  destroyed  by  heat  (60°  to  65°  C.) 


IMMUNOLOGY.      IMMUNITY  AND   IMMUNIZING   AGENTS 


243 


FIG.  60. — Illustrating  cell  receptors  of  the  first  order.  A  cell  receptor  (a)  uniting 
with  the  haptophore  (c)  of  the  toxin  molecule  or  antigen.  The  toxin  molecule  or  anti- 
gen consists  of  the  haptophore  and  the  toxophore.  The  toxophore  produces  the  toxic 
effects  upon  the  cell,  e  is  the  haptophore  of  the  cell  receptor  which  has  the  power  of 
combining  with  the  toxin  molecule  thus  neutralizing  its  possible  toxic  effects.  Free-cell 
receptors  constitute  the  antibodies,  and  are  ever  ready  to  combine  with  antigens  or 
toxins,  should  any  be  present.  Cell  receptors  and  antigen  bodies  are  specific  in  action. 
The  haptophore  of  the  diphtheria  cell  receptor  does  not  fit  the  haptophore  of  tetanus,  for 
example.  Each  antigen  or  toxin  reacts  with  the  antibodies  fitted  to  it.  (Journal  of 
ihe  American  Medical  Association,  1905,  p.  955.) 


r-/ 


FIG.  61. — Illustrating  receptors  of  the  second  order,  Fig.  54.  illustrating  receptors  of 
the  first  order,  c,  d,  The  cell  receptor  with  a  symop'hore  group  (d)  and  a  haptophore 
group  (e}  capable  of  combining  with  disintegrated  bacterial  substances  (/).  The  zymo- 
phore  group  produces  a  ferment  which  acts  upon  (disintegrates)  the  bacterial  cell  or 
blood-corpuscle,  as  the  case  may  be,  seized  upon  by  the  haptophore  group.  (Journal 
of  the  American  Medical  Association,  1905,  p.  1113.) 


244  PHARMACEUTICAL  BACTERIOLOGY 

while  the  haptophore  group  is  not  destroyed,  retaining  the  power  of  com- 
bining with  the  receptor  group  of  the  living  cell.  The  toxophore  group  is 
not  necessarily  simple.  It  may  comprise  two  or  more  different  groups. 
Snake  poison  contains  two  toxophore  groups,  one  agglutinating  red  blood 
cells,  the  other  causing  its  general  toxicity.  Diphtheria  toxin  also  has  two 
toxophore  groups,  the  one  causing  the  acute  symptoms  and  the  other,  the 
toxones  with  a  long  incubation,  causing  the  later  paralyses  and  cachexias. 

The  nature  of  immunity  to  these  antigens  is  conceived  as  follows :  The 
haptophore  group  is  bound  to  the  cell  receptor  because  of  a  specific  affinity. 
As  a  result  this  particular  side  chain  or  receptor  is  lost  to  the  living  cell  and, 
following  Weigert's  law  of  supercompensation  in  regeneration,  the  cell 
replaces  this  loss  by  producing  many  more  receptor  groups  than  were  pre- 
viously present.  As  in  the  callus  following  a  fracture  there  is  an  over- 
production. In  this  way  such  a  large  number  of  receptors  of  one  type  are 
produced  that  they  become  excessive  and  the  cell  thrusts  them  off  into  the 
blood  and  into  the  fluids  of  the  body.  Here  they  constitute  the  specific 
antibodies  and,  because  of  their  specific  affinity,  unite  with  the  haptophore 
group  of  toxins  and  prevent  their  reaching  the  cell  which  they  thus  protect. 

Therefore,  in  antitoxic  immunity  there  are  three  stages:  First,  the 
chemical  union  of  the  haptophore  group  of  antigen  to  the  receptor  group  of 
the  protoplasm  molecule;  second,  the  overproduction  and  liberation  of 
these  receptors  following  this  binding;'  and  third,  the  union  of  these  free 
receptors  or  antibodies  with  free  toxin  haptophore  groups  before  these  can 
reach  the  cells  to  injure  them  by  the  action  of  their  toxophore  groups.  The 
antigens  that  are  known  with  their  respective  antibodies  as  given  by 
Hektoen  are : 

Antigens  Products  of  Immunization 

Toxins — Antitoxins 

Ferments Antiferments 

Precipitinogens Precipitins 

Agglutinogens .  Agglutinins 

Opsonogens Opsonins 

Lysogens Amboceptors  or  lysins 

Antitoxins Antiantitoxins 

Agglutinins Antiagglutinins 

Complements Anticomplements 

Opsonins Antiopsonins 

Amboceptors Antiamboceptors 

Precipitins Antiprecipitins 

These  antibodies  all  result  from  the  overproduction  of  simple  receptors, 
but  the  protoplasm  of  cells  may  form  still  other  cell  receptors  which  are 
much  more  complicated  and  subserve  the  absorption  of  more  complicated 
and  complex  albuminous  molecules  than  those  of  toxins. 


IMMUNOLOGY.      IMMUNITY  AND    IMMUNIZING   AGENTS 


245 


Bacterial  clumping  or  agglutinating  phenomena  are  extremely  interest- 
ing as  well  as  valuable  in  the  diagnosis  of  disease.  Upon  this  behavior  of 
bacteria  depends  theWidal  typhoid  fever  test.  If  the  serum  of  an  animal 
inoculated  with  the  typhoid  bacillus  (antiserum)  is  added  to  a  liquid  cul- 
ture or  suspension  of  typhoid  bacilli,  the  latter  cease  to  move  and  after  a 
time  become  aggregated  into  irregular  clumps  or  masses.  The  same 
phenomenon  is  observed  if  instead  of  blood  of  a  typhoid  injected  animal,  the 
blood  of  a  typhoid  fever  patient  is  employed.  The  reaction  is  quite  specific, 


FIG.  62. — Illustrating  receptors  of  the  third  order,  or  so-called  amboceptors.  This 
serves  to  explain  the  action  of  lysins  (bacteriolysin,  hemolysin,  cell  lysins,  milk  lysins, 
etc.).  The  cell  receptor  (amboceptor)  has  two  haptophore  groups,  one  (e)  capable  of 
uniting  with  a  disintegrated  substance  as  bacterial  cell,  blood-corpuscle,  etc.,  (/)  and  the 
other  (g)  having  the  power  to  combine  with  a  complement  (&).  h  is  the  haptophore 
group  of  the  complement  (lysin)  and  3  the  zymotoxic  group.  Amboceptors,  lysin  re- 
ceptors and  receptors  of  the  third  order  mean  the  same  thing.  (Journal  of  the  American 
Medical  Association,  1905,  p.  1369.) 

^ 

though  not  absolutely  so.  That  is,  similar  agglutinating  phenomena  are 
produced  by  related  bacilli,  as  the  typhoid  bacillus,  the  para-typhoid 
bacillus  and  the  colon  bacillus.  Many  other  bacteria,  beside  the  colon- 
typhoid  group,  are  agglutinated  by  their  respective  antisera.  In  addi- 
tion to  diagnosing  disease  as  in  typhoid  fever  (the  Widal  test  gives  results 
even  before  there  are  marked  disease  symptoms),  the  agglutinating  phe- 
nomena are  useful  in  the  identification  of  bacteria.  The  technic  while  not 
difficult,  calls  for  many  precautionary  measures  and  requires  considerable 
time  and  care  to  avoid  erroneous  conclusions. 

In  1897  Kraus  found  that  when  the  germ-free  nitrates  from  broth  cul- 
tures of  bacteria  were  mixed  with  their  respective  antisera  (serum  from 
animals  inoculated  with  the  specific  bacteria)  the  formation  of  a  white 
precipitate  occurred.  The  substance  in  the  immunized  serum  which 
causes  the  formation  of  the  precipitate  has  been  termed  precipitin.  Simi- 


246  PHARMACEUTICAL  BACTERIOLOGY 

lar  reactions  are  observed  with  milk  and  egg  albumen,  when  used  with 
their  specific  immune  sera.  These  reactions  have  been  utilized  to  secure 
evidence  in  criminal  cases.  The  serum  of  an  animal  which  has  been  in- 
j  ected  with  human  blood  (humanized  immune  serum)  produces  a  precipitate 
when  mixed  with  human  blood,  even  in  high  dilutions.  Like  agglutina- 
tion, the  reaction  is,  however,  not  wholly  specific.  For  example,  human- 
ized animal  serum  will  also  produce  a  precipitate  with  the  blood  of  higher 
apes.  Dog  immunized  animal  serum  will  produce  a  precipitate  with  wolf's 
blood,  etc. 

The  chief  immunizing  agents  are  the  bacterolysins,  the  antitoxins  and 
the  leucocytes  (phagocytes)  aided  by  the  opsonins.  The  significance  of 
agglutinins  and  precipitins  in  the  prevention  of  bacterial  disease  is  not 
clear. 

Recent  observations  on  drug  action  tend  to  prove  that  some  of  these 
remedial  agents  apparently  possess  antitoxic  and  other  immunizing  prop- 
erties. It  is  for  example  fairly  well  proven  that  phosphorus  and  Echi- 
nacea  angustifolia  liave  the  power  of  increasing  the  opsonic  index  in 
certain  bacterial  invasions.  Sulphide  of  carbon  and  silica  appear  to  check 
and  cure  suppurative  processes,  perhaps  due  to  similar  activity.  Nuclein 
which  is  usually  derived  from  yeast,  is  reported  to  be  decidedly  bactericidal 
and  to  increase  phagocytosis  to  a  marked  degree.  According  to  Lloyd, 
Lobelia,  when  administered  hypodermically,  counteracts  the  toxin  of  the 
diphtheria  bacillus,  being  similar  in  its  effects  to  the  antidiphtheric  serum 
(antitoxin  of  diphtheria).  Belladonna  is  reported  to  be  prophylactic 
as  well  as  curative  in  scarlet  fever.  It  is  highly  probable  that  as  our  knowl- 
edge of  the  therapeutic  action  of  drugs  develops,  there  will  be  a  complete 
revolution  in  their  use  as  remedial  agents. 

2.  The  Immunizing  Agents. — The  following  is  a  brief  description  of 
the  more  important  immunizing  agents  of  the  body.  Other  immunizing 
agents  will  be  given  in  Chapters  XI  and  XII. 

a.  Phagocytosis. — As  already  explained  Metschnikoff  made  the  interest- 
ing discovery  that  the  white  blood  corpuscles  (leucocytes)  seized  upon  and 
disintegrated  and  fed  upon  bacteria  with  which  they  came  in  contact  or 
with  which  they  were  brought  in  contact.  Not  only  do  the  leucocytes 
possess  this  power  but  also  other  body  cells,  as  the  endothelial  cells  of 
capillaries,  and  the '  lymphocytes  which  are  cellular  structures  of  the 
lymph  channels  corresponding  to  and  developing  into  leucocytes  upon 
entering  the  circulation,  certain  fixed  body  cells,  more  especially  the  epithe- 
lial cells  of  the  intestinal  tract  and  of  mouth  and  throat  or  upper  respira- 
tory tract. 

The  germ  devouring  property  of  leucocytes  (phagocytosis  or  leucocy- 
tosis  of  Metschnikoff)  is  typically  illustrated  in  an  injury  to  the  skin, 


IMMUNOLOGY.      IMMUNITY   AND    IMMUNIZING    AGENTS  247 

as  cut,  abrasion  or  other  marked  injury.  In  a  more  or  less  severe  abrasion 
of  the  skin  there  is  a  tearing  of  the  tissues  as  epidermis,  derma  and  the 
lesser  arterioles  and  capillaries,  with  more  or  less  extra vasation_of_  red 
blood  corpuscles  and  serum.  Under  ordinary  conditions  such  a  wound 
is  always  infected,  primarily  and  principally  by  the  staphylococcus  group 
which  are  present  everywhere,  less  commonly  also  by  the  streptococcus 
group,  and  occasionally  by  the  tetanus  bacillus.  The  latter  being 
anaerobic  is  more  apt  to  develop  in  closed  wounds  or  deep  wounds.  It 
is  furthermore  a  spore  bearer.  These  organisms  find  the  serum  of  the 
blood  and  the  tissue  cell  juices  a  very  suitable  food  supply  and  an  active 
invasion  is  thus  set  up.  However,  the  body  defenders,  the  leucocytes,  are 
at  once  despatched  to  the  scene  of  action  to  repel  the  bacterial  invasion. 
After  the  hemorrhage  which  was  the  direct  result  of  the  mechanical  injury, 
has  ceased,  there  still  continues  an  exudation  of  blood  serum  carrying 
with  it  numerous  leucocytes,  and  these,  leucocytes  immediately  begin  the 
work  of  seizing  upon  and  devouring  the  invading  bacteria.  Vast  numbers 
of  the  leucocytes  are  killed  and  become  mixed  with  the  serum  exudate, 
with  broken  down  tissue  cells  and  with  bacteria,  constituting  the  pus. 
The  dying,  bacteria  gorged  leucocytes,  gradually  lose  the  power  of- 
ameboid  movement  and  finally  assume  a  fixed  spherical  form  and  consti- 
tute the  characteristic  pus  cells. 

Ordinarily  or  normally  the  leucocytes  gradually  gain  the  upper  hand, 
pus  becomes  more  and  more  scant,  finally  ceasing  to  form  altogether. 
Rejuvenescence  of  tissue  cells  begins  and  finally  the  damage  is  entirely 
repaired  and  the  wound  is  said  to  have  healed.  The  source  and  origin  of 
the  cells  concerned  in  regenerative  activities  is  as  yet  not  fully  deter- 
mined. It  is  known  that  in  the  case  of  the  infected  skin  injuries,  numerous 
leucocytes  and  lymphocytes  migrate  to  the  injured  area  and  are  largely 
concerned  in  phagocytic  activities,  but  these  body  cells  are  also  concerned 
in  the  healing  processes,  assisted  by  the  endothelial  cells.  These  several 
cellular  elements  constitute  the  so-called  inflammatory  lymph  which  enters 
into  the  formation  of  cicatricial  or  scar  tissue. 

The  older  writers  on  surgery  and  pathology  (fifty  to  sixty  years  ago) 
distinguished  between  "laudable  pus"  and  "sanious  pus."  It  was  be- 
lieved that  pus  formation  was  unavoidable  and  when  the  pus  was  of  a 
whitish  color  and  creamy  in  consistency  it  was  a  favorable  sign  as  indicat- 
ing a  "normal"  healing  process  and  such  pus  was  said  to  be  "laudable." 
On  the  other  hand,  if  the  pus  gradually  became  watery,  blood  tinged  and 
foul  smelling,  it  was  designated  "sanious"  and  the  wound  condition 
was  considered  unfavorable.  We  now  know  that  the  change  in  pus 
formation  designated  by  "sanious"  is  the  result  of  the  gain  of  the  in- 
vaders, the  pus  organisms,  more  especially  the  streptococcus  group. 


248  PHARMACEUTICAL  BACTERIOLOGY 

The  surgeons  of  today  seek  to  prevent  all  pus  formation.  All  surgical 
operations  are,  and  should  be,  without  infection  of  any  kind,  hence  there 
island  should  be,  no  pus  formation.  Cuts  or  incisions,  which  heal  without 
pus  formation,  are  said  to  show  "primary  union"  or  to  heal  by  "first 
intention,"  terms  which  were  in  constant  use  when  "Listerism"  was  ex- 
tensively introduced  into  surgical  practice  about  1875,  but  which  are  very 
rapidly  falling  into  disuse,  the  surgeons  of  today  simply  speaking  of 
infected  or  non-infected  wounds,  as  the  case  may  be. 

Occasionally  a  primary  invasion  by  the  staphylococcus  group,  {Staphy- 
lococcus  albus  (white  pus),  S.  aureus  and  S.  citreus  (yellow  pus),  Bacillus 
pyocyaneus  (blue  pus,  comparatively  rare) }  is  followed  by  an  invasion  by 
the  Streptococcus  which  may  result  in  a  more  or  less  widespread  infection 
commonly  known  as  "blood  poisoning,"  accompanied  by  redness,  swelling 
and  pain,  with  final  more  or  less  extended  tissue  destruction  and  necrosis. 

As  to  which  species  or  variety  of  infecting  microbe  initiates  the  primary 
infection  depends  upon  place  and  opportunity.  The  staphylococcus 
group  being  most  common  and  most  widespread,  naturally  is  most  likely 
to  be  the  predominating  infecting  agent.  We  may  expect  streptococcus 
infection  in  the  presence  of  abundant  decaying  organic  matter,  stable 
manure,  cattle  pens,  etc.  The  tetanus  infection  in  old  garden  soils,  in 
hay,  in  old  stable  manure  and  in  the  mud  and  dust  of  much  traveled  roads. 
For  example,  the  extensive  infection  of  wounds  by  the  tetanus  bacillus  in 
the  trenches  of  the  German- Allies'  battle  line  in  northern  France  (1915)  led 
to  the  suspicion  that  infected  bullets  had  been  used,  until  it  was  found 
that  the  old  garden  and  field  soils  of  this  region  were  the  prolific  sources 
of  the  infection. 

In  the  condition  known  as  boils,  acne,  carbuncles,  furuncles,  we  have 
active  infection  by  the  staphylococcus  and  streptococcus  groups  with  leu- 
cocytic  invasion  and  reaction.  There  may  be  infection  of  the  heart  (endo- 
carditis), of  the  joints  (rheumatoid),  of  the  pleurae,  of  the  kidneys  and^of 
other  organs,  especially  when  the  pus*  organisms  gain  access  to  the  circu- 
lation. An  extensive  local  infection  (as  in  marked  and  prolonged  tonsil- 
litis) may  result  in  the  infection  of  internal  organs,  often  with  serious  and 
even  fatal  results.  Localized  inflections,  as  pus  pockets  in  carious  teeth, 
bones,  mucous  tissues,  connective  tissues,  internal  organs,  etc.,  are  respon- 
sible for  rise  in  temperature  and  many  other  systemic  disturbances. 

Phagocytosis  is  also  marked  in  living  cells  outside  of  the  higher  animal 
organism.  For  instance  the  amebas  and  paramecia  are  active  and  even 
voraceous  devourers  of  bacteria  and  of  yeast  organisms.  It  has  even 
been  suggested  that  an  active  amebiasis  of  the  human  body  as  in  amebic 
dysentery  of  the  tropics  and  in  the  very  common  amebic  infection  of  the 
mouth  cavity  (pyorrhea,  Rigg's  disease),  the  infecting  organisms  act  pri- 


IMMUNOLOGY.      IMMUNITY   AND    IMMUNIZING    AGENTS  249 

marily  as  scavengers,  feeding  upon  the  multitude  of  bacteria  present. 
However,  that  may  be,  the  amebic  infection  does  induce  very  serious 
disturbances  which  were  difficult  to  cure  until  it  was  found  that-ipecac 
(emetin)  was  quickly  fatal  to  the  amebas  of  dysentery  as  well  as  to  those 
of  pyorrhea  (enteric  pills,  Lloyd  alcresta  ipecac  tablets;  or  hypodermic 
injections  of  the  active  principle  of  ipecac,  emetin). 

The  amebas  found  with  decaying  fruits  and  other  vegetable  matter 
feed  upon  bacteria  and  yeast  cells.  An  ameba  found  in  decaying  bananas 
limited  its  diet  almost  entirely  to  yeast  cells  which  it  disintegrated  very 
quickly. 

In  the  ordinary  conditon  of  phagocytosis  as  explained  under  staphy- 
lococcic  infection,  with  the  resultant  contra-invasion  by  the  white  blood 
corpuscles,  there  is  unquestionable  an  active  destruction  of  objectionable 
invaders  (the  bacteria),  brought  about  by  the  special  body  protectors  or 
guardians,  the  leucocytes,  through  the  assistance  of  the  opsonins,  and  such 
a  condition  may  be  designated  patrocytosis  as  contrasted  with  the  condi- 
tion as  we  find  it  in  amebic  dysentery  and  in  pyorrhea  where  the  relation- 
ship of  ameba  to  the  organism  invaded  (the  human  body)  is  harmful,  even 
though  the  amebas  feed  upon  body  bacteria.  In  malaria  for  example,  we 
have  true  parasitism.  The  invading  organism  (the  malarial  plasmodium) 
does  not  feed  upon  or  destroy  body  bacteria  and  does  not  render  even 
the  slightest  service  to  the  human  body. 

b.  Opsonins. — Opsonins  have  a  direct  relationship  to  leucocytosis  or 
phagocytosis  (patrocytosis).  Wright  and  others  proved  that  there  exists 
in  the  serum  of  the  blood  and  also  in  the  cells  of  the  body,  substances  which 
possessed  the  property  of  so  acting  upon  and  modifying  bacteria  as  to 
render  them  more  readily  seized  upon  and  devoured  by  the  leucocytes. 
Nothing  is  known  as  to  the  composition  of  these  substances.  No  one  has 
succeeded  in  isolating  them  in  a,  pure  state.  They  are  unquestionably 
proteid  in  nature  combined  with  the  serum  and  cell  proteids  of  the  body. 
They  are  specific  in  their  action.  That  is  an  opsonin  will  not  act  upon 
different  kinds  of  bacteria.  The  opsonin  which  acts  upon  the  Staphylo- 
coccus  aureus  so  preparing  these  organisms  as  to  make  them  more  readily 
seized  upon  and  digested  by  the  leucocytes,  will  not  act  upon  or  prepare 
the  organism  which  is  causative  of  pneumonia,  or  any  other  species  or 
variety  of  pathogenic  organism.  Each  species,  and  probably  also  each 
variety  of  pathogenic  microbe  is  acted  upon  by  a  special  or  specific 
opsonin. 

The  proof  of  the  existence  of  opsonins  is  as  follows:  If  fresh  blood 
is  mixed  with  an  emulsion  of  bacteria  and  incubated  at  body  temperature 
for  an  hour,  it  will  be  found  that  bacteria  are  within  the  white  blood  cor- 
puscles. If  we  wash  the  blood  corpuscles  free  from  serum  (by  means 


250  PHARMACEUTICAL  BACTERIOLOGY 

of  physiological  salt  solution  and  the  centrifugal  machine)  and  mix  the 
white  corpuscles  thus  removed  from  the  serum,  with  the  bacterial  emulsion 
and  incubate  as  before,  none  of  the  bacteria  will  be  found  within  the 
leucocyte.  This  test  however  merely  proves  that  the  blood  serum  influ- 
ences phagocytosis  or  leucocytosis.  In  order  to,  prove  that  the  action  is 
upon  the  bacteria  rather  than  upon  the  leucocytes,  the  following  test 
is  made.  Use  the  serum  free  leucocytes  as  before,  but  instead  of  adding 
the  plain  bacterial  suspension,  add  to  it  a  blood  serum  free  from  leucocytes. 
Mix  this  bacterial  suspension  now  containing  serum  with  the  leucocyte 
suspension,  incubate^  as  before  and  it  will  be  found  that  the  leucocytes 
again  contain  bacteria.  The  serum  has  in  some  way  acted  upon  (sensi- 
tized) the  bacteria  rendering  them  capable  of  being  seized  upon  by  the 
leucocytes.  The  phenomenon  may  be  compared  to  the  preparation  of 
food  by  cooking. 

Opsonins  are  specific  in  action  as  indicated,  are  thermostabile,  that  is 
they  are  not  destroyed  by  a  temperature  of  55°  C.  Quantitatively  they 
are  very  variable.  They  may  even  disappear  entirely  from  the  blood,  at 
least  temporarily.  Relative  quantitative  phagocytosis  indicates  what 
is  known  as  the  opsonic  index,  which  may  be  determined  as  follows: 
(Leishman's  method).  Mix  equal  parts  of  the  fresh  normal  blood  and  a 
bacterial  suspension  in  normal  salt  solution  and  incubate  for  30  minutes. 
Prepare  stained  slide  mounts  and  obtain  an  average  of  bacteria  per  leu- 
cocyte. This  will  give  the  normal  phagocytic  index,  given  as  i.  If, 
for  example,  the  normal  leucocyte  count  gives  an  average  of  ten  staphy- 
lococci  per  leucocyte,  and  a  comparative  count  of  a  patient  afflicted 
with  abscesses,  gives  an  average  of  2  staphylococci,  then  his  opsonic  index 
would  be  0.20,  that  is,  .much  below  normal. 

The  opsonic  index  may  be  raised  by  injecting  carefully  measured 
quantities  of  killed  cultures  (bacterins)  of  the  organisms  of  the  infection. 
However,  satisfactory  results  have  been  obtained  in  a  few  cases  only,  as 
in  acne,  in  pneumonia,  in  tubercular  infections,  and  in  a  few  other  infec- 
tions. Certain  bacterins  have  given  excellent  results  as  preventives,  as 
in  typhoid,  in  plague,  in  tetanus,  and  some  other  diseases. 

c.  Toxins,  Antigens,  Toxoids. — Toxins  are  poisonous  substances  elab- 
orated by  bacteria  and  other  pathogenic  organisms,  which  possess  the 
property  of  inducing  the  development  of  antitoxin  (an  ti- bodies,  immune 
bodies)  in  the  serum  of  the  blood  and  in  the  body  cells.  The  toxin  is  the 
result  of  the  activities  of  substances  formed  in  bacteria,  known  as  antigens, 
which  give  rise  to  the  anti-bodies  (antitoxins).  That  is,  the  antigen 
through  some  form  of  stimulation  gives  rise  to  substances  which  neutralize 
the  action  of  the  toxins  formed  by  the  toxigenic  bacteria.  Red  blood 
corpuscles  and  perhaps  also  other  body  cells  contain  antigens.  The  toxins 


IMMUNOLOGY.      IMMUNITY   AND    IMMUNIZING    AGENTS  251 

have  an  injurious  effect  upon  the  white  blood  corpuscles,  inhibiting  leu- 
cocytosis  (negative  chemo taxis).  If  virulent  cultures  of  the  bacillus  of 
anthrax  are  injected  into  susceptible  animals,  they  succumb  quickly- with- 
out any  evidence  of  leucocytosis  (negative  chemotaxis).  If  the  animal 
thus  injected  had  been  immunized  against  anthrax  by  means  of  atten- 
uated anthrax  cultures,  there  would  appear  large  numbers  of  leucocytes 
at  the  site  of  the  injection.  If  tetanus  bacilli  and  their  spores  are  washed 
free  from  toxin  and  injected,  active  leucocytoses  follows  (positive  chemo- 
taxis). The  interesting  observation  has  been  made  that  the  injection  of 
a  mixed  culture  of  highly  virulent  organisms  and  non-virulent  cultures 
(of  the  same  kind),  the  action  of  the  virulent  form  is  both  hastened  and 
increased.  It  is  suggested  in  explanation  that  the  leucocytes  preferably 
seize  upon  the  non- virulent  forms  and  have  as  a  result  little  energy  left 
to  seize  upon  the  virulent  forms. 

Toxins,  therefore,  are  intimately  concerned  in  the  processes  of  immuni- 
zation. They  induce  the  development  of  anti-bodies  which  overcome 
or  neutralize  the  very  substances  -elaborated  by  the  pathogenic  bacteria 
and  they  have  a  markedly  checking,  retarding  or  inhibiting  in- 
fluence on  leucocytosis.  This  apparently  contradictory  action  of  toxins 
is  not  as  yet;  satisfactorily  explained.  Metchnikoff  suggested  that  immune 
bodies  as  well  as  complement  were  enzyma  tic  in  nature  and  that  both  were 
produced  by  the  leucocytes,  thus  doing  away  with  the  necessity  for  assum- 
ing that  the  immune  bodies,  (anti-bodies),  were  the  result  of  the  action  of 
the  toxins  or  antigens.  However,  it  cannot  be  denied  that  the  anti-bodies 
are  the  result  of  the  presence  and  influence  of  the  antigens  or  toxins. 

The  toxins  or  poisons  elaborated  by  certain  animals,  as  poisonous 
snakes,  resemble  the  antigens  of  bacteria  in  that  they  are  capable  of 
inducing  the  formation  of  anti-bodies.  The  following  animal  anti- 
toxins are  now  on  the  market:  Antivenene  for  rattlesnake  poisoning, 
anti- toxin  for  scorpion  poisoning;  and  anti- toxins  for  eel,  fish,  turtle,  wasp 
and  salamander  poisons.  Most  of  the  animal  toxins  are  not  of  a  simple 
molecular  structure.  The  toxin  molecule  of  rattlesnake  venom,  for 
example,  has  a  distinct  toxophore  group  which  give  rise  to  the  general  toxic 
symptoms,  and  a  hemolytic  group  which  disintegrates  the  red  blood  cor- 
puscles, and  the  two  act  quite  independently  of  each  other. 

True  toxins  are  formed  by  certain  higher  plants,  as  ricin  by  the  caster 
bean  plant,  crotin  by  the  croton  bean  plant,  robin  by  the  locust,  abrin  by 
the  jequerity  bean  plant,  and  pollenin  by  certain  members  of  the  composite 
family  (golden  rods,  rag  weeds,  and  others).  These  several  toxins  when 
introduced  into  the  body  will  give  rise  to  anti-bodies  which  are  used  to 
overcome  or  neutralize  the  specific  toxins  (anti-ricin,  anti-abrin,  anti-robin, 
anti-crotin,  and  anti-pollenin). 


252  PHARMACEUTICAL  BACTERIOLOGY 

It  is  known  that  tissue  cells  react  more  or  less  specifically  with  bacterial 
toxins  and  these  reactions  are  used  for  making  certain  diagnostic  tests, 
such  as  the  skin  reaction  tests  for  tuberculosis,  glanders,  syphilis,  diph- 
theria and  typhoid  fever. 

d.  Antitoxins. — The  antitoxins  are  the  substances  found  in  the  body 
cells  and  in  the  blood  plasm  which  neutralize  or  destroy  the  toxins  -and 
toxoids  of  pathogenic  bacteria.     As  has  already  been  explained,  when  a 
pathogenic  organism  is  introduced  into  the  body,  the  lysins  destroy  the 
cell  membrane,  setting  free  the  endotoxins  which  by  their  presence  stimu- 
late the  formation  of  the  antitoxins.     The  antitoxins  are  specific  in  nature. 
That  is,  the  antitoxin  against  the  diphtheric  endotoxin  is  not  active 
against  the  toxin  of  typhoid,  or  of  plague. 

e.  Anti-antitoxins. — It   is  known  that  the  normal  bodily  resistance 
to  disease  varies  from  time  .to  time  and  that  the  artificial  immunity  due 
to  the  introduction  of  specific  antibodies  gradually  wanes  and  finally 
disappears.     This  variability  in  the  action  of  antibodies  is  supposed  to 
be  due  to  substances,  again  specific  in  character,  in  the  body  cells  and  in 
the  blood  plasm,  which  destroy  the  antibodies. 

/.  Agglutinins.—  Agglutinins  are  specific  cellular  prqducts  which  cause 
bacteria  to  clump  or  gather  into  groups,  preceded  by  a  cessation  in  motion 
in  those  organisms  which  possess  motility.  The/Widal  typhoid  fever 
test  is  based  upon  this  phenomenon. 

Agglutinins  are  produced  artificially .  by  injecting  bacteria  into  the 
circulation  of  various  animals.  The  serum  of  such  animals  contains  the 
bodies  which  give  rise  to  the  agglutinating  phenomena.  Sera  can  be 
so  highly  agglutinative  as  to  produce  this  reaction  in  dilutions  of  1-100,000 
or  more.  In  typhoid  patients  the  agglutinins  generally  appear  after 
the  fifth  day  and  may  persist  for  a  long  time  (several  years)  after  con- 
valescence. By  some  it  is  supposed  that  the  phenomenon  of  agglutination 
is  a  preliminary  stage  in  the  development  of  lysins.  It  is  however  a  fact, 
that  while  the  bactericidal  action  of  serum  is  destroyed  at  a  temperature 
of  s6°C.,  the  agglutinating  power  survives  until  62°C.  is  reached.  Bacteri- 
cidal sera  do  not  interfere  with  the  agglutinating  power.  These  and  other 
observations  appear  to  indicate  that  the  agglutinins  are  specific  bodies 
independent  of  the  other  immunizing  agents. 

g.  Precipitins. — Precipitins  are  specific  bodies  which  occur  in  the 
blood  of  an  immunized  animal,  as  rabbit  or  guinea  pig.  If,  for  example, 
a  rabbit  is  immunized  against  human  blood  (through  repeated  injections 
of  human  blood  directly  into  the  circulation  of  the  animal)  and  the  serum 
of  such  immunized  blood  be  mixed  with  a  trace  of  human  blood,  a  precipi- 
tate is  formed.  The  reaction  is  strictly  specific,  excepting  that  the  blood 
immunized  against  a  sheep  will  form  precipitate  with  the  blood  from  the 


IMMUNOLOGY.      IMMUNITY  AND   IMMUNIZING   AGENTS  253 

goat.  Blood  immunized  against  the  ape  will  form  precipitate  with 
human  blood.  The  test  has  been  used  practically  in  medico-legal  and  in 
criminal  cases.  The  details  for  making  the  test  may  be  found  in  the 
larger  works  on  bacteriology,  parasitology  and  on  immunization.  The 
method  is  given  in  full  in  Bacteriological  Methods  in  Food  and  Drug 
Laboratories.  P.  Blakiston's  Son  and  Company.  1915.  (^Schneider.) 

h.  Virulency — Aggressins. — The  pathogenic  bacteria  form  substances 
which  protect  against  the  defensive  measure  of  the  host,  however,  one 
and  the  same  species  of  pathogenic  or  toxigenic  organism  is  not  at  all  times 
equally  active  defensively.  In  other  words,  the  virulency  of  bacteria  is 
variable.  In  the  manufacture  of  diphtheria  antitoxin  it  is  desirable  to 
use  a  strain  of  the  causative  organism  which  is  highly  virulent,  in  order  to 
hasten  as  well  as  to  increase  the  formation  within  the  blood  of  the  horse, 
of  the  defensive  or  protective  bodies.  The  virulency  may  be  lowered  or 
weakened  ( attenuated)  in  various  ways.  Exposure  to  high  temperatures 
may  accomplish  this.  In  fact  the  high  bodily  temperature  in  fevers  is 
nature's  method  of  reducing  the  virulency  of  the  invading  organism. 
Again,  the  virulency  may  be  lowered  and  completely  modified  qualitatively 
by  a  change  of  host,  as  in  cow  pox.  An  organism  maybe  entirely  harmless 
and  even  highly  useful  in  one  position,  and  become  highly  virulent  in 
another  position  in  the  same  organism.  Thus  the  B.  coll  is  a  normal  and 
beneficent  inhabitant  of  the  intestinal  tract;  but  should  it  be  introduced 
(accidentally  or  by  design)  into  the  peritoneal  cavity  or  into  any  tissue 
other  thari  the  intestinal  tract,  it  may  set  up  serious  abscess  formation. 
The  virulency  of  the  causative  agent  of  rabies  is  lowered  as  well  as  modi- 
fied by  exposure  to  dry  air.  In  a  general  way  all  agencies  which  lower  the 
vitality  of  pathogenic  and  toxigenic  organisms  tend  to  lower  the  virulency, 
although  there  are  some  notable  exceptions. 

As  it  is  possible  to  lower  (attenuate)  as  well  as  to  increase  (augment) 
the  activity  (virulency)  of  objectionable  microorganisms,  just  so  is  it 
possible  to  increase  the  activity  of  useful  organisms.  Thus  the  free  nitro- 
gen assimilating  power  of  the  Rhizobia  group  may  be  greatly  increased 
by  growing  the  organisms  upon  special  media,  or  we  may  reduce  this 
power  greatly  or  practically  destroy  it.  To  such  changes  in  beneficent 
organisms  we  apply  the  term  potency,  rather  than  virulency.  We  in- 
crease or  augment  the  potency  of  a  yeast  ferment,  and  on  the  other  hand 
we  increase  or  augment  the  virulency  of  the  diphtheria  germ. 

The  subject  of  increased  virulency  of  pathogenic  organisms  has  re- 
ceived a  great  deal  of  attention  within  recent  years.  Bail  and  others 
made  some  interesting  observations  which  may  be  summarized  as  follows : 
By  injecting  pure  cultures  of  tubercle  bacilli  into  the  peritoneal  cavity 
of  a  guinea  pig,  a  rapidly  fatal  tuberculosis  is  produced.  If  the  peritoneal 


254  PHARMACEUTICAL   BACTERIOLOGY 

exudate  from  this  guinea  pig  is  sterilized  and  injected  into  a  second 
guinea  pig,  together  with  some  tubercle  bacilli  of  the  kind  used  in  the 
first  animal,  the  second  animal  will  succumb  much  more  rapidly  than  the 
first,  usually  within  twenty-four  hours.  If  the  sterilized  exudate  alone 
is  injected,  nothing  will  happen;  and  if  the  tubercle  bacilli  alone  are 
injected  the  tuberculosis  will  develop  within  a  few  weeks,  as  in  the  first 
animal.  It  is  assumed  that  the  peritoneal  exudate  contains  a  substance 
formed  by  the  tubercle  bacilli  which  has  the  power  of  greatly  increasing 
the  virulency  and  this  substance  has  been  called  aggressin.  Heating  the 
exudate  to  6o°C.  causes  a  further  increase  in  the  virulency  of  the  aggressin, 
and  it  was  found  that  small  amounts  of  the  exudate  were  relatively  more 
virulent  than  larger  amounts,  and  Bail  assumes  that  there  are  two  sub- 
stances in  the  exudate,  one  which  is  thermolabile  preventing  rapid  death, 
the  other  thermostabile,  favoring  rapid  death.  It  is  assumed  that  a 
bacterolysin  is  formed,  which  acting  on  the  bacilli,  liberates  the  endotoxin 
which  paralyzes  the  polynuclear  leucocytes,  the  mononuclear  phagocy- 
tosis being  undiminished. 

i.  Anaphylactic  Reactions. — Anaphylaxis  is  the  opposite  of  prophy- 
laxis. It  indicates  a  state  of  susceptibility  or  rather  hypersusceptibility 
to  certain  substances  when  brought  in  biological  contact  with  living 
cells.  The  term  was  originally  used  to  indicate  a  condition  of  hyper- 
susceptibility  to  diseases  generally.  More  recently  the  term  anaphylaxis 
or  anaphylactic  shock,  has  been  very  largely  applied  to  the  hypersus- 
ceptibility toward  horse  serum.  The  investigations  into  anaphylaxis 
were  prompted  by  the  comparatively  common  cases  of  serum  sickness 
(rash,  urticarias,  enteritis,  etc.)  and  the  comparatively  rare  sudden  col- 
lapse and  death,  following  the  use  of  diphtheria  antitoxin.  These  classic 
investigations  proved  that  the  anaphylactic  reaction,  or  state,  depended 
upon  the  following  essentials: 

First. — Injecting  into  the  experimental  animal  (as  rabbit  or  guinea 
pig)  a  dose  of  some  non-toxic  protein,  which  sensitizes  the  animal  specifi- 
cally to  this  particular  substance. 

Second. — An  incubation  period  of  from  eight  to  fourteen  days,  followed 
by 

Third. — A  second  injection  of  the  same  protein,  at  the  close  of  the 
incubation  period.  The  anaphylactic  reaction  appeared  almost  im- 
mediately (collapse  and  death). 

Fourth. — The  sensitization  developed  by  the  initial  dose  may  endure 
for  months  and  even  for  years,  in  fact  may  endure  for  life  and  may  be 
transmitted  to  the  offspring. 

The  experiments  also  proved  that  the  anaphylactic  condition  can 
be  developed  toward  a  great  variety  of  substances,  animal,  vegetable  and 


IMMUNOLOGY.      IMMUNITY   AND    IMMUNIZING    AGENTS  255 

even  mineral.  The  sensitization  or  anaphylactic  reaction  phenomena 
are  extremely  variable  in  kinds  as  well  as  in  degree.  The  following  is  a 
brief  summary  of  the  subject: 

Disease  is  nothing  more  nor  less  than  an  anaphylactic  reaction  to  the 
proteins  of  the  causative  agents,  as  bacteria  and  protozoa.  The  reactions 
indicate  that  nature  is  endeavoring  to  overcome  the  toxins  formed,  de- 
veloped or  generated.  It  is  presumed  that  no  one  will  take  a  disease 
unless  first  sensitized  to  the  specific  causative  agent.  What  is  generally 
known  as  the  incubation  period  in  disease  corresponds  to  the  sensitiza- 
tion period  in  anaphylaxis.  Recovery  from  disease  simply  means  that  the 
characteristic  reaction  (as  manifested  by  the  symptoms)  has  been  suc- 
cessful. If  death  is  the  outcome,  it  means  that  the  reaction  was  exhausted, 
paralyzed  or  broken  down. 

Hypersusceptibility  to  certain  foods,  more  especially  to  roe,  fish, 
shellfish,  eggs,  milk,  cheese,  rhubarb,  strawberries,  tomatoes  and  cereals, 
is  fairly  common.  The  symptoms  usually  appear  soon  after  eating  and 
vary  in  kind  as  well  as  in  degree,  depending  upon  the  nature  of  the  food 
and  the  degree  of  susceptibility  to  it.  There  may  be  urticarias,  erythemas, 
prickly  heat,  spasms,  asthmatic  conditions,  respiratory  difficulties, 
fever,  gastrointestinal  disturbances  and  occasionally  sudden  and  complete 
collapse.  Physicians  have  noted  that  eczema  is  frequently  caused  by 
certain  foods,  as  excess  of  fats  and  starch.  The  so-called  cyclical 
vomiting  of  children  is  supposed  to  have  its  origin  in  food  susceptibility, 
associated  perhaps  with  an  inherited  neurasthenic  condition. 

It  is  theoretically  suggested  that  man  was  originally  hypersensitive 
to  all  foods  and  as  a  result  of  the  anaphylactic  reaction  the  specific  anti- 
bodies or  protective  bodies  were  gradually  developed,  finally  establishing 
full  prophylaxis  toward  those  substances  which  we  now  recognize  as 
harmless  and  wholesome  foods.  The  reason  why  we  cannot  use  the 
deadly  night-shade,  or  nux  vomica,  or  aconite,  or  tobacco,  as  foods  is 
because  we  are  still  in  a  state  of  hypersusceptibility  toward  these 
plants.  There  are  many  marked  racial  differences  in  food  hypersuscep- 
tibility. The  goat  and  the  hog  will  thrive  on  vegetables  which  are  highly 
toxic  to  man.  The  existence  of  an  anaphylactic  reaction  toward  a 
given  substance,  as  strychnine,  aconite,  curara,  etc.,  indicates  an  effort 
to  establish  immunity.  If  the  amount  of  ingested  food  substance  for 
which  immunity  is  not  yet  established  is  excessive,  the  reaction  may  be 
wholly  exhausted  with  disastrous  results.  Anaphylaxis  is  nature's 
warning  against  over-indulgence  in  a  food  for  which  prophylaxis  is 
not  yet  fully  established. 

Anaphylaxis  which  is  developed  parenterally,  that  is  by  bringing 
the  reacting  protein  or  other  substance  in  direct  physiological  contact 


256  PHARMACEUTICAL  BACTERIOLOGY 

with  body  cells,  without  exposure  to  enteral  enzymatic  action,  is  quanti- 
tatively as  well  as  qualitatively  different  from  the  anaphylactic  conditions 
following  the  administration  of  the  reacting  substances  per  os.  The 
fluids  and  enzymes  of  the  digestive  tract  modify  profoundly  the  anaphy- 
lactic reaction.  It  is  true,  similar  enzymes  exist  within  the  general  body 
cells,  but  here  they  are  not  associated  with  activating  elements.  The 
enteral  solutions  and  enzymes  break  down  (catalyze)  many,  in  fact  most, 
of  the  organic  compounds,  rendering  them  anaphylactically  harmless. 

Some  very  interesting  observations  have  been  made  in  regard  to 
anaphylactic  skin  reactions  with  food  substances.  If  a  trace  of  a  given 
food  substance  or  an  aqueous  extract  thereof  is  briskly  rubbed  into  a 
small  skin  abrasion,  hypersusceptibility  or  anaphylaxis  toward  the  food 
will  be  indicated  by  circumscribed  redness  which  develops  quickly  and 
disappears  in  a  short  time.  The  redness  (inflammatory  process)  is 
simply  a  local  anaphylactic  reaction.  Such  tests  have  been  made  with  a 
great  variety  of  raw,  partially  cooked  and  completely  cooked  foods.  The 
skin  reaction  is  simply  a  localized  anaphylactic  shock.  If  the  same  sub- 
stance were  injected  into  the  general  circulation,  a  general  bodily  reaction 
might  follow. 

Skin  reaction  tests  along  the  lines  above  outlined  could  no  doubt  be 
made  of  great  practical  value  to  the  diagnostician,  the  clinician,  in  forensic 
medicine,  in  criminal  investigation,  in  the  study  of  dietetics,  in  food  in- 
vestigation, etc.  It  would  be  possible  to  determine  hypersusceptibility, 
not  only  to  foods,  but  to  a  variety  of  other  substances.  The  physician 
makes  use  of  a  number  of  recognized  skin  reactions,  as  in  typhoid  fever, 
in  diphtheria,  in  tuberculosis,  in  glanders,  in  syphilis,  in  gonorrhea,  in 
hay  fever.  Not  only  may  the  existence  of  the  disease  be  ascertained  by 
such  tests,  but  the  degree  of  susceptibility  to  such  diseases  may  be  as- 
certained. 'This  latter  information  would  prove  of  great  value  as  in- 
dicating the  diseases  against  which  any  existing  hypersusceptibility 
should  be  overcome  by  appropriate  means,  as  perhaps  avoiding  as  much 
as  possible  the  chances  of  exposure  to  such  diseases.  Tests  determining 
the  susceptibility  to  drug  action  would  be  a  means  of  selecting  proper 
dosage.  In  fact,  experiments  with  non-toxic  plant  extracts  administered 
parenterally  is  opening  up  a  new  system  of  therapeutic  activity.  The 
essentials  in  this  newer  therapy  are  based  upon  the  assumption  that  certain 
wholly  non- toxic  plant  substances  which  are  enzymatic  in  nature,  as 
chlorophyll,  lipoids  and  vitamines,  act  biologically  in  splitting  up  foreign 
proteins,  as  bacteria,  pathological  deposits  and  formations  as  in  cancer, 
in  goiter,  in  syphilis,  in  gout,  in  rheumatism,  etc.  It  is  claimed  that 
excellent  results  have  already  been  obtained  from  the  intravenous  and 
intramuscular  administration  of  plant  extracts  and  metallic  colloids 


IMMUNOLOGY.      IMMUNITY  AND   IMMUNIZING   AGENTS 


257 


in  cancer,  in  goiter,  in  chronic  eczema,  in  tuberculosis,  in  hay  fever  and  in 
other  intractable  diseases. 

An  animal  (as  rabbit  or  guinea  pig)  which  has  been  sensitized  to  any 
specific  substance,  as  human  blood,  deer's  blood,  egg  albumin,  casein, 
cow's  milk,  goat's  milk,  horse  serum,  specific  plant  extracts,  poisons 
of  many  kinds,  etc.,  will  show  a  skin  reaction  toward  the  specific  substance 
for  which  it  was  sensitized.  One  and  the  same  animal  may  in  fact  be 
sensitized  to  three  or  more  substances  at  the  same  time  and  will  show  a 
separate  and  distinct  and  specific  reaction  for  each  substance.  The 
specificity  fails  or  rather  merges  or  blends  with  many  closely  related 
substances.  Thus  a  guinea  pig  sensitized  to  human  blood  will  react 
toward  the  blood  of  the  ape.  Other  interacting  sensitizations  develop 
toward  rat  and  mouse,  dog  and  wolf,  horse  and  ass,  sheep  and  goat. 
Organ  specificity  is  also  highly  interesting  and  gives  some  apparently 
contradictory  results.  For  instance,  a  guinea  pig  sensitized  to  cerebral 
extract  of  a  rabbit  will  react  toward  the  cerebral  extract  of  widely  dif- 
ferent species,  but  will  not  react  toward  other  tissues. 

It  may  indeed  be  possible  to  sensitize  one  animal  toward  a  large 
number  of  substances  and  such  animal  may  then  be  employed  for  making 
skin  reaction  tests  with  any  one  or  all  of  the  substances  in  question. 
Thus  the  physician  could  use  this  animal  for  diagnostic  purposes;  the 
criminologist  to  ascertain  whether  or  not  a  given  blood  stain  was  of 
human  origin;  the  food  analyst  to  determine  whether  or  not  the  albumen 
is  from  duck's  eggs  or  from  hen's  eggs;  the  pathologist  for  the  purpose 
of  ascertaining  the  malignancy  or  benignancy  of  a  tumor;  the  bacteri- 
ologist to  determine  the  group  relationship  of  a  given  microorganism;  the 
toxicologist  to  ascertain  the  identity  of  a  poison,  etc. 

Bacteriolysins. 
Opsonins. 
Natural      J  Phagocytes. 

I  Glandular  secretions 


Immunizing 
Agents 


Active 


Inherited  or  normal 


Augmented — Immunizing  diseases. 


Artificial 


Modified  toxins  and  un- 


Toxins. 

Small-pox     vaccina- 


modified  toxins. 
Bacterial — Bacterins  or  vaccines. 


tion. 
Rabies  vaccination. 


Passive 


[  Sera — Diphtheric,  tetanic,  glandular  extracts,  etc. 
Drugs — Nuclein,  lobelia,  phosphorus,  etc. 


17 


CHAPTER  XI 

SEROLOGY— THE    MANUFACTURE    AND   USE   OF   SERA   AND 

VACCINES 

The  most  wonderful  recent  discoveries  in  the  science  of  bacteriology 
pertain  to  the  relationship  of  pathogenic  germs  and  the  serum  of  the  blood 
of  susceptible  animals.  As  already  stated  blood  serum  has  bactericidal 
properties  (see  lysins),  but  it  is  often  not  sufficiently  active  to  destroy  cer- 
tain invading  germs  (pathogenic)  and  the  disease  manifestations,  due  to 
the  toxins  liberated  by  the  germs,  gradually  develop.  The  bacterial  toxins 
are  of  two  kinds,  those  which  escape  from  the  bacterial  cells  and  are  soluble 
in  the  surrounding  media,  entering  the  system  by  absorption;  and  those 
which  remain  within  the  germ  cell  and  are  set  free  only  on  the  breaking 
up  of  the  bacterial  cells.  The  former  are  the  toxins  proper  or  exotoxins, 
the  latter  are  called  endotoxins.  As  already  explained  the  toxins  cause  the 
development  within  the  serum  of  the  blood  of  certain  substances  (anti- 
bodies), which  neutralize  or  overcome  the  effects  of  the  toxins  and  which 
are  called  antitoxins.  Investigators  hoped  that  experiments  would  prove 
that  every  pathogenic  germ  would  cause  the  development  of  a  correspond- 
ing antitoxin  which  might  be  used  in  the  treatment  of  the  disease.  This 
hope  has  not  been  realized.  Of  the  numerous  experimentations  with  anti- 
toxins only  one  has  thus  far  proven  entirely  satisfactory,  namely,  the  anti- 
toxin of  diphtheria.  Several  others  have  proven  more  or  less  useful,  as 
will  be  explained  later,  but  they  are  far  from  satisfactory. 

The  antitoxins  act  by  neutralizing  the  bacterial  toxins  of  the  disease, 
and  not  by  acting  upon  and  killing  the  germs  themselves.  In  this  regard 
the  antitoxins  or  antitoxic  sera  differ  from  the  antibacterial  or  bactericidal 
sera,  which  act  by  preventing  the  development  of  the  bacteria.  This 
distinction  and  difference  is  not  generally  understood.  The  bactericidal 
sera  have,  however,  thus  far  proven  quite  unsatisfactory  in  the  treatment 
of  disease.  They  are  not  standardized  by  units  as  are  the  antitoxins. 
The  dose  is  by  volume,  from  10  to  50  cc.,  and  even  more,  usually  given 
hypodermically.  The  sera  are  produced  by  injecting  toxins  or  the  toxic 
germs  (artificially  cultured)  into  the  animal,  as  the  horse.  As  a  rule  the 
first  injections  consist  of  dead  germs;  finally,  living  germs  of  different 
virulency  may  be  used.  By  this  means  a  tolerance  is  established.  The 
serum  obtained  from  animals  thus  immunized  is  used  in  the  treatment  of 
disease,  its  action  depending  upon  its  bactericidal  properties.  There  is  a 

258 


SEROLOGY— MANUFACTURE    AND    USE    OF    SERA    AND    VACCINES       259 

group  of  sera  known  as  composite,  which  give  evidence  of  being  a  decided 
improvement  over  the  simple  sera.  They  are  called  composite  because 
they  have  the  peculiar  qualities  of  two  distinct  forms  of  immunity^— for 
example,  diphtheria-immune  horses  may  be  used  in  the  subsequent  bac- 
terial inoculation,  which  gives  the  resulting  immune-serum  a  double  con- 
tent of  a  corresponding  antibacterial  body  and  of  diphtheric  antitoxin. 
This  subject  is  as  yet  entirely  in  the  experimental  stage.  It  is  also  known 
that  one  kind  or  type  of  immunity  has  some  influence  not  only  upon  other 
immunities,  but  also  upon  other  diseases.  The  antitoxin  of  diphtheria, 
for  example,  appears  to  act  as  a  cure  or  prophylactic  against  pathological 
conditions  other  than  diphtheria. 

We  now  come  to  a  third  class  of  substances  used  in  the  treatment  of 
disease,  namely,  the  bacterial  vaccines,  also  designated  bacterins  and 
opsonogens  (Ohlmacher).  The  term  vaccine  (from.  Vacca,  a  cow)  is 
appropriately  applicable  to  the  small-pox  remedy,  but  is  entirely  inappli- 
cable to  these  newer  agents.  Either  bacterin  or  opsonogen  is  a  suitable 
name. 

Bacterins  are  simply  suspensions  of  dead  pathogenic  germs  which  are 
used  in  the  treatment  of  disease.  A  homologous  or  autogenous  bacterin 
is  prepared  from  germs  taken  direct  from  the  patient  and  is  used  in  treat- 
ing the  same  patient.  A  heterologous  bacterin  is  one  which  is  derived 
from  a  source  other  than  the  patient  under  treatment.  A  mixed  bacterin 
is  one  in  which  the  germs  (of  the  same  species)  used  are  derived  from 
several  sources.  The  manufactured  bacterins  (heterologous)  ready  for 
use  by  the  physician  are  called  stock  vaccines  or  stock  bacterins. 

The  following  is  a  tabulation  of  antitoxins,  toxins,  antibacterial  sera 
and  bacterins  found  upon  the  market  and  used  by  physicians  and 
veterinarians. 

i.   For  Human  Use 

A.  Antitoxic  Sera  or  Antitoxins. 

Antidiphtheric  serum. 

Liquid  or  usual  form. 

Concentrated  form. 

Dry  form  (official  in  some  pharmacopoeias). 
Antitetanic  serum. 

Liquid  or  usual  form. 

Dry  form. 

B.  Antibacterial  Sera  or  Bactericidal  Sera. 

Antistreptococcic  serum. 
Antipneumococcic  serum . 
Antimeningitic  serum. 


260  PHARMACEUTICAL  BACTERIOLOGY 

Antityphoid  serum. 

Antidysenteric  serum. 

Antigonorrheal  serum. 

Antiplague  serum  (Yersin's  serum). 

Antianthrax  serum. 

Scarlet  fever  serum  (Marpmann's  serum). 

Antituberculous  serum  (anti tuberculins). 

C.  Bacterins  or  Opsonogens.     (Vaccines.)     (Homologous  or  autogen- 
ous, heterogenous  and  mixed.) 
Staphylococcus. 

S.  pyogenes  albeus. 


S.  pyogenes  aureus. 


used  singly  or  mixed. 


S.  pyogenes  citreus. 
Streptococcus. 
Gonococcus. 
Typhoid. 

Typhoid  (Shafer's  mixed  bacterin). 
Cclon  bacillus. 
Neoformans  bacillus. 
Pyocyaneous  bacillus. 

Bubonic  plague  bacillus  (Haffkine's  plague  vaccine). 
Tuberculins. 

Tuberculin,  old  (T.  O.). 

Tuberculin  residuum  (T.  R.). 

Tuberculin  precipitate  (T.  P.). 

Bacillus  emulsion  (B.  E.). 

Bacillus  nitrate  (B.  F.). 

D.  Toxins  (modified). 
Small-pox  vaccine. 
On  ivory  points. 
In  glycerinated  tubes. 
Dry  form. 

Hydrophobia  vaccine. 
Erysipelas  and  prodigiosus  toxin.     (Cancer  and  other  malignant 

growths.) 
Antivenine.     (Snake  toxin.) 

2.  For  Veterinary  Use 
A.  Antitoxic  Sera  or  Antitoxins. 
Antitetanic  serum. 
Influenza  serum.     (Intravenous  use.) 
Hog  cholera  serum. 


SERO1.OGY — MANUFACTURE    AND    USE    OF    SERA    AND    VACCINES      261 

B.  Antibacterial  Sera  or  Bactericidal  Sera. 

Antistreptococcic  serum. 
Canine  distemper  serum. 
White  scour  serum. 

C.  Bacterins. 

Anthrax. 

Mallein. 

Tuberculin. 

Blackleg. 

Blacklegine. 

Blacklegules  (pill  form).  * 

Blacklegoids  (pill  form).  < 

Hog  cholera. 

Fowl  cholera. 

White  scour. 

Texas  fever. 

The  above  substances  resemble  each  other  in  that  they  are  organic  and 
of  complex  chemical  composition.  They  gradually  deteriorate  and  finally 
become  worthless,  some  sooner  than  others.  Even  the  comparatively  per- 
manent kinds  will  not  retain  their  full  activities  more  than  a  few  months, 
though  they  may  still  be  sufficiently  active  therapeutically  after  eighteen 
months,  or  even  longer.  They  should  be  kept  in  a  cool  dry  place,  away 
from  light.  Turbidity  in  those  preparations,  which  are  clear  when  freshly 
prepared,  indicates  that  decomposition  changes  have  set  in  and  that  they 
are  unfit  for  use.  Many  of  the  bacterins  are  normally  turbid  and  nearly 
all  of  them  have  some  slight  color  and  odor. 

Thus  far  only  a  few  of  the  substances  above  tabulated  have  proven 
entirely  satisfactory  in  the  treatment  of  the  particular  disease  or  diseases 
for  which  they  are  intended.  This  is  but  to  be  expected  since  their  use  is 
very  largely  based  upon  theory.  Theory  and  practice  have  ever  failed  to 
develop  along  exactly  parallel  lines.  Science  is  however  fortunate  in 
being  able  to  assert  that  in  the  antidiphtheric  serum  we  have  practically  a 
specific  for  the  cure  of  diphtheria,  provided  it  is  used  in  time  and  given  in 
sufficiently  large  and  sufficiently  frequent  doses.  The  antitetanic  serum 
has  given  excellent  results  particularly  as  a  preventive,  as  has  also  the  anti- 
streptococcic  serum.  Of  the  bacterins  the  Staphylococcus  has  given  ex- 
cellent results  in  the  cure  of  actual  pathologic  conditions.  Some  of  the 
others  have  proven  less  satisfactory  and  in  many  cases  their  great  useful- 
ness lies  in  their  preventive  rather  than  curative  powers. 

We  will  explain  very  briefly  the  manufacture  of  a  few  of  these  sub- 
stances only,  as  the  methods  are  quite  closely  similar  for  like  agents.  The 


262  PHARMACEUTICAL   BACTERIOLOGY 

following  is  a  brief  outline  of  the  manufacture  of  the  marvelous  remedy  for 
the  treatment  of  the  dread  disease  of  childhood,  namely  diphtheria. 

i.  Antidiphtheric  Serum 

A.  Selecting  and  Testing  the  Horse. — Ordinary,  normal,  non-pedigree 
horses  are  preferred,  purchased  under  a  guarantee  of  soundness.     Even 
though  purchased  under  such  a  guarantee  the  animal  is  kept  under  ob- 
servation for  a  few  weeks  and  tested  for  glanders  by  the  mallein  test. 
No  animal  is  retained  until  it  is  proven  that  there  is  no  latent  or  active 
disease  present.     The  animal  is  well  housed  and  well  cared  for  during  the 
entire  time,  under  conditions  as  sanitary  as  it  is  possible  to  make  them. 
All  laboratories  are  also  regularly  visited  by  a  U.  S.   Government  in- 
spector, who  reports  his  findings  to  Washington. 

B.  Preparing  the  Toxin  of  Diphtheria. — Pure  cultures  of  a  selected 
strain  of  the  diphtheria  bacillus,  possessed  of  a  high  potency,  virulency  or 
toxicity,  are  made  in  liter  flasks  containing  beef  bouillon.     The  original 
bacilli  thus  used  are  taken  from  some  patient  suffering  with  diphtheria,  and 
by  means  of  isolation  methods  all  foreign  microbes  are  rejected  or  excluded. 
After  the  culture  is  several  days  old  or  when  a  maximum  amount  of  the 
toxin  has  been  formed  and  deposited  in  the  bouillon,  the  bacili  are  killed  by 
adding  0.25  per  cent,  of  trikresol.     The  bouillon  with  the  dead  bacilli  is 
filtered.     The  clear  filtered  substance  constitutes  the  toxin  which  is  in- 
jected into  the  horse  for  the  purpose  of  developing  (in  the  horse)  the  anti- 
toxin of  diphtheria.     The  virulency  or  potency  of  the  toxin  varies  and  is 
tested  on  guinea-pigs  and  compared  with  the  U.  S.  Government  standard. 
The  highiy  toxic  race  or  strain  of  germs  is  perpetuated  in  the  laboratory 
by  daily  transfers  to  new  culture  tubes.     In  this  manner  the  bacilli  are 
maintained  for  a  long  time,   several  years  or  longer.     However,   even 
with  the  greatest  care  the  race  finally  deteriorates,  weakens  or  undergoes  a 
change  in  potency  and  it  becomes  necessary  to  secure  a  new  stock  culture. 

C.  Developing  the  Antitoxin  of  Diphtheria  in  the  Horse. — Twice  weekly 
the  horse  is  given  (by  hypodermic  injection  into  the  flank  region)  gradually 
increasing  doses  of  the  toxin  of  diphtheria.     The  rule  is  to  give  enough  to 
produce  a  marked  reaction.     For  a  day  or  two  the  horse  is  sick  with  diph- 
theria, then  recovers  as  the  increased  antitoxin  in  the  blood  (serum)  of  the 
animal  neutralizes  the  toxin.     This  is  continued  for  from  four  to  six  weeks 
when  a  maximum  amount  of  antitoxin  has  presumably  developed.     The 
last  dose  of  toxin  is  several  hundred  times  greater  than  the  first. 

D.  Bleeding  the  Horse. — A  sterilized  canula  or  trochar  is  inserted  into 
the  jugular  vein,  after  the  neck  has  been  thoroughly  washed  with  soap  and 
water,  shaved  and  rinsed  with  a  5  per  cent,  solution  of  carbolic  acid.     The 
blood  is  drawn  off  into  sterilized  liter  tubes,  which  are  plugged  with  cotton. 


SEROLOGY — MANUFACTURE    AND    USE    OF    SERA    AND    VACCINES       263 

From  nine  to  twelve  liter  of  blood  are  taken  from  the  horse  at  one  time  and 
the  bleeding  is  repeated  four  or  five  times  at  intervals  of  about  six  months. 
The  punctured  wound  is  closed  by  keeping  an  artery  forceps  in  position 
for  a  short  time. 

E.  Securing  the  Serum. — The  blood  tubes  are  set  aside  until  the  clot 
has  formed  and  settled  to  the  bottom.  The  clear  serum  is  siphoned  off 
into  a  large  flask,  0.25  per  cent,  of  trikresol  is  added  as  a  preservative  and 


FIG.  63. — Bleeding  the  horse  after  a  maximum  amount  ot  the  antitoxin  of  diph- 
theria has  been  developed  in  the  blood.  The  animals  pay  but  little  attention  to  the 
operation. 

to  kill  any  germs  that  might  be  accidentally  present,  and  then  filtered 
through  several  thicknesses  of  filter  paper,  under  pressure  (suction).  The 
perfectly  clear,  sterile  and  germ-free  serum  constitutes  the  antitoxin  of 
diphtheria  and  is  ready  for  use  as  soon  as  it  is  standardized  and  put  into 
suitable  containers. 

F.  Standardizing  the  Antitoxin  of  Diphtheria. — Since  the  antitoxic  va- 
lence of  horse  serum  as  above  described  varies  somewhat,  it  is  necessary  to 
determine  the  quantitative  value  in  order  that  physicians  may  know  what 
amounts  to  administer  in  the  treatment  of  diphtheria.  The  standard 
unit  of  strength  now  adopted  by  all  civilized  countries  is  the  so-called 


264 


PHARMACEUTICAL  BACTERIOLOGY 


Ehrlich  unit,  which  is  the  amount  of  serum  (antitoxin  of  the  horse)'  which 
will  just  neutralize  one  hundred  times  a  fatal  dose  of  toxin  when  adminis- 
tered to  a  guinea-pig,  weighing  approximately  250  grams  or  one-half  pound. 
The  U.  S.  standard  is  prepared  in  the  biological  laboratories  of  the  U.  S. 
Public  Health  Service  at  Washington,  and  every  manufacturer  of  diph- 


PIG.  64. — Sterilized  liter  tubes  into  which  the  blood  drawn  from  the  horse  is  placed. 
The  top,  covered  with  sterilized  cloth,  is  connected  with  the  canula  in  the  jugular  vein 
of  the  animal. 

theric  antitoxin  in  the  United  States  is  supplied  with  standard  units  from 
this  laboratory.  The  method  of  procedure  is  approximately  as  follows : 
Eight  containers  (test-tubes)  are  set  out  in  a  row  and  numbered  or  marked 
serially.  Into  each  tube  is  poured  just  one  hundred  fatal  doses  of  toxin 
(fatal  to  a  250  gram  guinea-pig,  determined  experimentally),  and  a  graded 
amount  of  the  serum  to  be  standardized,  so  that  the  first  tube  has,  in  all 


SEROLOGY — MANUFACTURE    AND    USE    OF    SERA    AND    VACCINES      265 

probability,  not  enough  antitoxin  to  neutralize  the  one  hundred  fatal  doses 
of  the  toxin,  and  the  eighth  tube  has,  in  all  probability,  a  great  excess  of 
antitoxin.  The  contents  of  one  tube  is  injected  into  a  guinea-pig,  thus 
requiring  eight  pigs.  The  animals  are  marked  and  kept  under  close 
observation.  The  first,  second  and  perhaps  third  die,  showing  that  not 
enough  serum  was  added  to  neutralize  the  toxin.  The  fourth  pig  just 
recovers,  showing  that  the  amount  of  serum  added  to  the  fourth  tube 
was  sufficient  to  neutralize  one  hundred  fatal  doses  of  the  toxin.  This 


FIG.  65. — Guinea-pigs  in  wire  cages.  These  lively  little  animals  are  used  in  test- 
ing the  virulense  of  the  diphtheria  toxin  which  is  injected  into  the  horse  and  also  for 
the  purpose  of  standardizing  the  antitoxin.  The  reasons  why  these  animals  are  pre- 
ferred are  wholly  biological  and  physiological.  They  propagate  rapidly,  are  easily 
kept,  easily  handled,  and  respond  (biologically)  to  the  tests  applied. 


amount  of  serum  (antitoxic)  represents  one  unit.  From  this  amount 
or  unit  the  quantities  to  be  put  into  the  containers  are  determined.  500, 
1000,  2500  and  5000  unit  quantities  are  put  up,  for  the  convenience  of 
physicians.  500  to  1000  units  constitute  an  immunizing  dose,  given  to 
those  who  do  not  have  diphtheria,  but  who  have  been  exposed  to  the 
disease.  The  larger  doses  are  curative.  The  rule  is  to  give  large  doses, 
repeated  as  often  as  may  be  necessary.  1000  units  are  usually  em- 
ployed as  immunizing  doses;  3000,  5000  and  10,000  unit  packages  for 
curative  doses. 


266 


PHARMACEUTICAL   BACTERIOLOGY 


2.  Concentrated  Diphtheric  Antitoxin 

While  the  chemical  nature  of  antitoxin  is  not  known,  it  has  been  deter- 
mined that  it  is  united,  in  some  way,  with  the  globulins  of  the  blood.  The 
attempts  to  isolate  antitoxin  have  resulted  in  the  manufacture  of  a  refined 
or  concentrated  antidiphtheric  serum  which  is  used  quite  extensively 


LJ 


FIG.  66.  —  Container  with  diphtheria  antitoxin,  supplied  with  hypodermic  needle, 
piston,  all  ready  for  immediate  use  by  the  physician.  The  plunger  is  simply  a  homeo- 
pathic vial  with  rubber  stopper.  (Cutler  Laboratory.) 


though  it  does  not  meet  with  the  unqualified  favor  accorded  the  antidiph- 
theric serum.     The  process  of  manufacture  is  as  follows  : 

a.  The  antidiphtheric  serum  is  saturated  with  amBioftwm  sulphate 


which  precipitates  the  globulins  (containing  the  antitoxin)  in  the  form  of  a 
white  mass.     It  is  then  filtered  and  the  filtrate  rejected. 

b.  The  precipitate  left  on  the  filter  is  redissolved  in  water  and  this  solu- 
tion is  again  treated  with  ammonium  sulphate  as  in  (a).  The  object  in 
redissolving  in  water  is  to  wash  the  globulins. 


SEROLOGY — MANUFACTURE    AND    USE    OF    SERA    AND    VACCINES      267 

c.  The   second  precipitation  product  is  treated  with  a  saturated  salt 
solution  which  dissolves  the  antitoxin  globulins.  The  solution  is  then  filtered. 

d.  To  the  filtered  solution  2.5  per  cent,  of  acetic  acid  is  added  which 
again  precipitates  the  globulins  on  the  filter  paper  where  it  is  partially 
dried  by  means  of  filter  paper  and  towels  pressed  upon  the  mass. 

e.  The  partially  dried  material  is  placed  in  a  dialyzing  bag  and  sus- 
pended in  a  water  current,  for  several  days.     This  removed  the  salts  by 
osmotic  action  and  at  the  same   time  the  globulins  enter  into  solution 
within  the  bag. 

f .  A  preservative  is  added  to  the  liquid  which  is  then  passed  through 
a  Berkefeld  filter.     Some  physiologic  salt  solution  is  also  added.     This  is 
the  final  product. 

g.  After  being  tested  bacteriologically  to  make  sure  that  it  is  not  con- 
taminated, it  is  standardized  as  described  under  diphtheric  serum. 

The  above  process  removes  the  following  non-active  substances: 
serum  albumins,  lecithin,  cholesterin,  traces  of  bile  salts  and  acids,  blood 
salts  and  the  non-antitoxic  globulins.  The  dosage  of  the  concentrated 
antitoxin  is  less  than  that  of  the  non-concentrated  serum  and  it  keeps 
longer.  For  the  manufacture  of  the  concentrated  diphtheria  antitoxin 
the  returned  serum  is  generally  employed,  that  is  serum  which  has  ex- 
ceeded the  time  limit  of  use. 

3.  Antitetanic  Serum 

This  is  prepared  similarly  to  antidiphtheric  serum.  The  tetanus 
bacilli  are  grown  in  bouillon,  in  the  absence  of  oxygen,  since  tetanus  germs - 
are  anaerobic.  The  growth  is  then  killed,  filtered  out  and  the  clear  toxic, 
germ-free  bouillon  filtrate  is  utilized  in  the  immunization  of  the  horse. 
Small  doses,  usually  mixed  with  some  antitetanic  serum,  are  administered 
at  first  and  gradually  increasing  the  amount  as  the  horse  can  stand  it  until 
large  quantities  are  given,  even  as  much  as  700  or  800  cc.  After  some 
months  the  horse  is  bled  in  the  same  manner  as  for  antidiphtheric  serum, 
the  serum  is  separated  and  bacteriologically  tested  in  the  same  way. 

The  unit  of  tetanus  antitoxin  is  that  quantity  of  antitetanic  serum 
which  is  necessary  to  completely  neutralize  1000  fatal  doses  of  tetanus 
toxin  for  a  250-gram  guinea-pig. 

Antitetanic  serum  has  not  been  a  marked  success  as  a  curative  agent. 
Its  greatest  usefulness  appears  to  be  as  a  prophylactic,  for  which  purpose  it 
should  be  given  early,  as  soon  as  the  injury  (cut,  gunshot  wound,  abrasion) 
has  occurred. 

The  following  are  the  more  important  antibacterial  sera.  A  fuller 
description  of  the  processes  of  manufacture  is  omitted  as  that  is  a  matter  df 
no  special  importance  to  the  pharmacist.  Furthermore,  manufacturers  do 
not,  as  a  rule,  disclose  full  details  of  manufacture. 


PHARMACEUTICAL   BACTERIOLOGY 
4.  Antipneumococcic   Serum 

This  serum  is  obtained  from  horses  immunized  against  the  Pneumo- 
coccus  and  is  employed  in  the  treatment  of  pneumonia  and  other  infectious 
disease  in  which  this  germ  is  present.  The  dose  is  about  10  cc.  repeated 
several  times  a  day,  given  h\*podermically.  The  serum  must  be  kept  in  a 
cool  dark  place.  \Vhen  a  tube  is  opened  the  contents  should  be  used  with- 
in twenty-four  hours,  sealed  temporarily  with  sealing  wax,  paraffin  or 
sterile  wadding.  This  serum  has  not  proven  very  satisfactory,  though  it 
is  safe  and  worthy  of  a  trial.  (See  pneumonia.) 

5.  Antoneningococcic   Serum 

Antimeningococcic  serum  is  obtained  from  horses  which  have  been 
immunized  with  cultures  of  Diplococcus  meningitidis  intraccllularis,  be- 
ginning with  dead  cultures,  then  using  living  cultures  and  finally  with 
autolvsate.  Its  use  is  said  to  have  met  with  considerable  success  in  the 
treatment  of  cerebro-spinal  meningitis,  when  injected  into  the  spinal 
canal  in  doses  of  10  cc.,  repeated  dairy.  The  serum  acts  as  an  antitoxin, 
it  increases  phagocytosis  and  also  acts  as  a  bactericide.  It  should  be 
used  early  in  the  course  of  the  disease. 

6.  Yersm's  Serum   (Antiplague  Serum) 

Yersin?s  serum  is  made  by  injecting  horses,  first  with  dead  plague 
bacillus  cultures  (Bacillus  pestis)  and  finally  with  the  living  organisms.  It 
has  been  used  with  varying  success  in  plague  epidemics.  Large  doses 
(30  to  50  cc.)  should  be  administered  (h\ixxiermically)  early  in  the  course 
of  the  disease.  Its  chief  value  is,  however,  prophylactic.  The  liquid 
form  of  the  serum  may  also  be  used  for  intravenous  injection.  The  dry 
serum  is  said  to  keep  indefinitely  and  must  be  dissolved  before  using. 

7.  Bacterins 

a.  Ordinary  Bacterins. — The  bacterins  are  still,  so  to  speak,  on  trial. 
Some  have  given  excellent  results  while  others  are  wholly  unsatisfactory. 
The  preference  appears  to  be  for  autogenous  bacterins.  The  majority  of 
physicians  are,  however,  compelled  to  use  the  so-called  stock  bacterins, 
or  the  manufactured  bacterins  ready  for  use,  for  the  reason  that  few  phv- 
sicians  have  the  time  or  the  equipment  to  prepare  the  homologous  or  auto- 
genous bacterins.  The  method  of  preparing  a  homologous  bacterin  may 
be  outlined  as  follows: 

a.  A  tube,  flask  or  plate  with  the  suitable  culture  medium  (agar  or 
gelatin)  is  inoculated  with  the  germs  taken  from  the  patient  and  incubated, 
until  a  maximum  development  has  taken  place,  about  twenty-four  hours. 

b.  The  growth  is  separated  from  the  culture  medium  by  means  of  a 


SEROLOGY — MANUFACTURE    AND    USE    OF    SERA    AND   VACOHES      269 

sterile  physiological  salt  solution  and  a  platinum  wire  loop.  The  salt 
solution  with  the  bacteria  is  transferred  to  a  sterile  test-tube  which  is  then 
sealed  in  a  flame. 

c.  When  the  tube  is  cool,  it  is  shaken  vigorously  so  as  to  emulsify  the 
bacteria  in  the  salt  solution. 

d.  The  tube  is  opened  and  about  one  drop  is  removed  with  which  to 
make  the  blood-corpuscle  count,  to  be  explained  later.    The  tube  is  again 
sealed  in  the  flame. 

e.  The  tube  is  now  placed  hi  a  water  bath  (opsonic  incubator  of  spedal 
construction  for  this  work)  at  a  temperature  of  60°  C.  for  a  sufficient  length 
of  time  to  kill  the  germs;  one  hour  is  usually  adequate.    This  constitutes 
the  bacterin  and  is  ready  for  use  as  soon  as  it  is  standardized.    Usually 
some  preservative  is  added  when  the  tube  is  opened  and  before  the  bacterin 
is  injected  (0.2  per  cent,  lysol,  0.4  per  cent,  trikresol,  etc.). 

f .  From  the  above  it  must  be  evident  that  no  two  preparations  contain 
the  same  number  of  germs  per  cc.  and  hence  the  physician  cannot  know 
how  many  dead  microbes  are  injected  at  a  dose.    Therefore  the  necessity 
of  standardizing  the  bacterin,  which  is  done  as  follows: 

g.  Mix  one  part  of  freshly  drawn  blood  with  one  part  of  the  bacterin 
(taken  from  the  tube  in  d.),  add  two  or  three  parts  of  physiological  salt 
solution,  and  spread  evenly  on  a  slide.     Examine  under  the  microscope  and 
determine  the  number  of  microbes  per  cc,  in  terms  of  the  number  of  red 
blood-corpuscles  per  cc.     This  is  done  by  making  numerous  (10  to  20) 
counts  of  red  blood-corpuscles  and  microbes.     Knowing  that  there  are 
5,000,000,000  red  blood-corpuscles  per  cc.,  it  is  then  a  simple  matter  to 
compute  the  number  of  microbes  per  cc.  in  the  bacterin  under  considera- 
tion.    The  count  thus  determined  divided  by  the  number  of  bacteria 
desired  for  one  dose,  indicates  the  number  of  times  the  bacterin  is  to  be 
diluted.     This  is  very  clearly  illustrated  in  a  chart  prepared  by  Houghton. 
shown  in  Fig.  67. 

The  number  of  bacteria  administered  per  dose  depends  upon  the  thera- 
peutic effects  to  be  produced,  the  kind  of  bacterin  used,  the  nature  of  the 
disease  and  the  condition  of  the  patient.  The  rule  is  to  start  with  small 
doses,  gradually  increasing  them  in  such  a  manner  as  to  secure  a  maximum 
of  positive  opsonic  phases  with  a  minimum  of  negative  opsonic  phases. 
In  round  numbers  the  dosage  ranges  from  5,000,000  to  50,000,000  bacilli, 
represented  by  varying  quantities  of  the  bacterins. 

b.  Sensitized  Bacterins. — The  ordinary  bacterins  effect  protection 
against  disease  in  two  ways.  a.  Stimulating  the  formation  in  the  body 
cells  and  in  the  blood  serum,  the  specific  amboceptors  which  wffl  increase 
phagocytosis,  b.  Stimulating  the  development  of  the  specific  antibodies 
which  will  neutralize  the  specific  bacterial  toxin  (endotoxin).  It  takes 


270 


PHARMACEUTICAL   BACTERIOLOGY 


from  five  to  ten  days  to  develop  the  immunizing  effects,  and  their  use  was 
accompanied  by\more  or  less  severe  local  as  well  as  systemic  reactions 


PIG.  67. — Counting  the  bacteria  in  standardizing  bacterins.  This  chart  shows  the 
count  of  bacteria  and  of  red  blood  cells  in  twenty  successive  fields  of  the  microscope. 
The  number  of  red  cells  counted  (308)  is  to  the  number  of  bacteria  counted  (224)  as  the 
number  of  red  cells  per  cubic  centimeter  in  normal  blood  (5,000,000,000)  is  to  the  number 
of  bacteria  per  cc.  in  the  suspension  (3,636,000).  This  count  (3,636,000)  divided  by  the 
count  desired  in  the  final  dilution(4OO, 000,000)  gives  the  number  of  times  (9)  this  sus- 
pension must  be  diluted  to  bring  it  to  the  desired  dilution.  (Parke,  Davis  &  Co.). 

;      .  > 

(anaphylaxis) .     These  undesirable  qualities  are  said  to  be  avoided  or  over- 
come by  the  use  of  the  so-called  sensitized  bacterins  or  sero-bacterins. 


SEROLOGY — MANUFACTURE    AND    USE    OF    SERA    AND    VACCINES       271 


The  sensitized  bacterins  differ  from  the  ordinary  bacterins  in  that  they 
are  pre-charged  or  saturated  with  the  specific  amboceptors,  thus  when 
injected  hypodermically  or  intravenously,  the  body  is  at  once  stimulated 
to  form  the  specific  antibodies  (anti-endotoxins) .  They  are  prepared  as 
follows,  using  typhoid  sero-bacterin  as  an  example.  Twenty-four  hour 
pure  cultures  of  the  Bacillus  typhosus  are  placed  in  normal  salt  solution 
(0.85  per  cent.).  The  mixture  is  thoroughly  emulsified  and  filtered  into  a 
centrifuge  tube  and  to  it  is  added  immune  goat's  serum  (that  is,  blood 
from  a  goat  which  has  been  immunized  against  typhoid)  and  let  stand  for 
twenty-four  hours  at  a  temperature  of  24°  C.,  with  frequent  shaking. 
Saline  solution  is  added,  shaken,  and  centrifuged  for  5-6  minutes.  The 
supernatant  liquid  (saline  solution  containing  most  of  the  excess  of  im- 
mune serum)  is  drawn  off.  More  saline  is  added,  shaken  and  again  cen- 
trifuged, and  the  supernatant  liquid  again  drawn  off  (saline  solution 
containing  the  last  trace  of  the  excess  of  immune  serum) .  The  reason  why 
the  excess  immune  serum  must  be  drawn  off  is  because  experience  has 
demonstrated  that  it  would  interfere  with  the  development  of  the  active 
immunization  by  the  bacterial  antigen.  The  bacteria  left  in  the  cylinder 
of  the  centrifuge  are  now  said  to  be  sensitized  by  the  immune  serum  and 
constitute  the  so-called  sensitized  bacterin  or  sero-bacterin,  or  in  this  par- 
ticular case,  the  typho-serobacterin.  Some  bacteriologists  are  of  the 
opinion  that  the.  living  sensitized  bacteria  should  be  used,  whereas  others 
are  of  the  opinion  that  the  dead  bacteria  are  just  as  effective  and  their 
use  is  not  accompanied  by  the  possibility  of  spreading  active  infection, 
although  this  is  not  likely  as  far  as  the  typho-serobacterin  is  concerned. 
(This  organism  developing  in  the  intestinal  tract  and  not  hypodermically.) 
It  would  appear  that  the  present  tendency  is  to  prefer  the  killed  bacteria. 
For  the  purpose  of  killing  the  sensitized  bacteria,  heat  or  phenol,  or  other 
antiseptic,  is  use.  The  bacteria  are  then  counted  by  the  Wright  methods 
(explained  elsewhere)  and  standardized  suspensions  are  made  for  use  as 
a  preventive  of  typhoid  and  also  as  a  cure.  At  the  present  time  it  is 
customary  to  inject  a  trivalent  typho-serobacterin,  consisting  of  the  sen- 
sitized Bacillus  typhosus  and  of  Bacillus  paratyphosus  A  and  of  B.  para- 
typhosus  B.  Definite  numbers  of  the  sensitized  bacteria  are  injected  at  a 
dose,  from  125,000,000  to  2,000,000,000,  suitably  suspended  in  saline 
solution.  Most  of  the  sensitized  bacterins  are  used  for  the  purpose  of 
developing  active  immunizations  rather  than  as  cures,  although  some  of 
them  have  proven  quite  effective  in  certain  chronic  stages  of  disease. 
The  sero-bacterins  possess  the  following  properties  and  advantages  over 
the  ordinary  bacterins. 

i.  They  produce  quick  active  and  lasting  immunity,  which  begins 
within  24  to  48  hours  after  they  are  introduced  into  the  system. 


272  PHARMACEUTICAL  BACTERIOLOGY 

2.  There  is  no  negative  phase  (opsonic)  as  in  the  use  of  the  ordinary 
bacterins.     The  negative  phase  (represented  by  aggravation  of  symptoms 
and  reduction  in  phagocytosis)  following  the  use  of  the  old  bacterins  is  due 
to  the  fact  that  the  bacteria  take  up  specific  antibodies  (amboceptors) 
from  the  blood  of  the  patient. 

3.  As  compared  with  the  ordinary  bacterins,  the  local  reaction  (pain, 
congestion,  erythema,  etc.)  are  greatly  reduced,  and  the  general  or  sys- 
temic reaction  (fever,  headache,  etc.)  is  practically  eliminated. 

The  work  on  sensitized  bacterins  is  recent  and  much  of  the  research 
which  led  to  the  present  perfection  of  these  preparations  must  be  credited 
to  Besredka,  Ehrlich,  Metchmkoff,  Gay,  Theobald  Smith,  Gordo,  Meyer 
and  Babes.  Sero-bacterins  have  been  extensively  tried  out  during  the 
world  war,  in  nearly  all  of  the  armies,  particularly  the  trivalent  typho- 
sero-bacterin.  As  the  result  of  the  use  of  this  remedial  agent  typhoid 
fever  has  become  non-existent  in  the  army,  once  the  deadliest  foe.  The 
U.S.  army  statistics  show  that  the  typhoid  case  rate  has  fallen  from  3.03 
per  thousand  in  1909  to  0.009  per  thousand  in  1914  (or  a  reduction  of 
98  per  cent.);  and  the  mortality  rate  fell  from  0.28  per  thousand  in  1909 
to  o  in  1913.  Certainly  a  convincing  showing. 

The  following  are  the  more  important  sero-bacterins  now  in  use. 

Acneic.  Autogenous  or  polyvalent  stock  preparation.  Used  in 
acute  as  well  as  in  chronic  cases. 

Asiatic  Cholera. — Univalent.     For  preventive  immunization. 

Bacillus  coli. — Univalent,  autogenous,  or  polyvalent  (strains).  In 
fistula,  local  infections,  catarrhal  jaundice. 

Influenza. — Polyvalent.     In  influenza,  catarrh,  colds.     Prophylactic. 

Gonococcic. — In  chronic  cases. 

Meningococcic. — Preventive  immunization. 

Pertussic. — Bacillus  pertussis.     Preventive  and  as  a  cure. 

Plague. — Active  and  rapid  immunization. 

Pneumococcic. — Univalent  and  polyvalent. 

Pyorrheic. — Polyvalent.     In  bacterial  pyorrhea. 

Staphylococcic. — Autogenous   and  polyvalent.     Treatment. 

Streptococcic. — Treatment  of  erysipelas,  infections,  abscesses. 

Typhoid. — Univalent  and  trivalent.     Immunization  and  treatment. 

8.  Tuberculins 

The  tuberculins  are  of  special  interest  as  they  give  great  promise  in  the 
successful  treatment  of  tuberculosis.  The  different  kinds  have  their 
special  use.  Their  manufacture  is  briefly  outlined  as  follows: 

A.  Tuberculin  Old  (T.  O.). — This  is  the  original  Koch  tuberculin  or 
Koch  lymph  and  is  a  concentrated  bouillon  culture  of  the  tubercle  bacillus, 


SEROLOGY — MANUFACTURE    AND    USE    OF    SERA    AND    VACCINES      273 

which  has  been  filtered  to  remove  the  germs.     It  is  a  toxin  solution  and 
not  a  bacterin  proper. 

B.  Tuberculin  Residuum  (T.  R.). — This  is  prepared  by  grindihg~the 
dried  tubercle  bacilli,  extracting  with  water,  centrifugalizing,  discarding 
the  supernatant  liquid,  regrinding  the  sediment,  which  is  first  allowed  to 
dry,  and  mixing  with  glycerin  and  water.     It  is  thus  a  suspension  of 
pulverized  tubercle  bacilli  in  an  aqueous  solution  of  glycerin.     The  grind- 
ing  process   is   tedious   and   requires   much   time.     The    tuberculin   is 
standardized  so  that  i  cc.  will  represent  10  mg.  of  the  dry  culture. 

The  supernatant  liquid,  after  centrifugalizing,  is  sometimes  drawn  off, 
instead  of  rejecting,  and  constitutes  the  upper  tuberculin  (T.  O.  )  (Obere 
Tuberculin).  These  two  tuberculins  (the  T.  R.  and  the  T.  O.)  differ  in 
therapeutic  value  and  in  physical  properties. 

C.  Bacillus  Emulsion  (B.  E.). — This  consists  of  pulverized  tubercle 
bacilli  suspended  in  50  per  cent,  glycerin  and  is  standardized  to  contain 
5  mg.  of  solid  matter  per  cc.      It  differs  from  T.  R.  in  that  the  supernatant 
liquid  (T.  O.)  is  not  drawn  off. 

D.  Tuberculin  Precipitate  (T.  R.). — This  is  obtained  from  old  tuber- 
culin by  precipitation  with  alcohol,  drying  and  pulverizing  the  precipitate. 
It  is  used  in  making  the  Calmette  eye-test.     (See  tuberculosis). 

E.  Bouillon  Filtrate     (Tuberculin  Filtrate  B.  F.— Denys  Tuberculin). 
The  tubercle  bacillus  cultures  are  passed  through  a  Berkefeld  filter  to 
remove  'all  germs.     The  filtrate  is  preserved  with  trikresol. 

9.  Small -pox  Vaccine 

Small-pox  vaccine  is  not  a  true  toxin  nor  yet  a  true  bacterin.  Its 
value  in  the  eradication  of  small-pox  has  world-wide  recognition.  The 
following  is  the  manner  in  which  small-pox  vaccine  is  prepared. 

a.  Selecting  the  Animal. — A  young  heifer  (five  to  ten  months  old)  is 
selected,  tested  for  tuberculosis  by  means  of  tuberculin.     The  animal  is 
observed  for  a  time  to  make  sure  of  general  condition  of  health;  is  well  fed 
and  well  cared  for,  under  conditions  as  sanitary  as  it  is  possible  to  keep 
them. 

b.  Inoculating  the  Animal. — The  heifer  is  strapped  securely  to  a  frame- 
work, back  down,  the  udder  region  is  cleansed,  shaven  and  cross  marked 
(scarified)  with  a  sharp  scalpel.     The  cuts  are  just  deep  enough  to  cause 
the  escape  of  serum,  not  actual  bleeding.     This  scarified  surface  is  then 
inoculated  with  glycerinated  small-pox  virus  taken  from  a  patient.     When 
the  inoculated  material  has  had  time  to  be  absorbed  the  animal  is  righted 
again   and  cared  for  under  as  aseptic  conditions  as  possible.     In  time 
(six  to  seven  days)  pustules  form  over  the  entire  inoculated  area.     The 
virulent   virus   from  man  conveys  the  disease  to  the  animal,  but  in  its 

18 


'74 


PHARMACEUTICAL   BACTERIOLOGY 


passage   through    the  animal  it  becomes  modified,  losing  in  viruk 
yet  capable  of  producing  immunity  as  the  result  of  a  mild  intoxication 
(vaccinia). 

c.  Remaning  the  Scab. — The  animal  is  again  fastened  to  the  frame. 
The  inoculated  surface  is  washed  and  dried.  The  thick  scab  which  has 
formed  over  the  inoculated  area  is  then  removed  and  triturated  with  50  per 
cent,  glycerin.  This  constitutes  the  small-pox  vaccine. 


PIG.  6&. — Thortaj,  the  heifer  Oiapyed  to  the  frame,  preparatory  to  removing  the 
which  was  scarified  and  inoculated  with  the  small- pox  virus. 

••ft 

d.  Aging  or  Ripening  the  Vaccine. — The  fresh  or  raw  vaccine  is  not 
used  as  it  contains  various  living  microbes.    It  is  acted  upon  by  the 
glycerin  added,  for  five  or  six  weeks.     The  virus  is  tested  bacteriologically 
during  this  period,  and  as  soon  as  no  more  colonies  appear  it  is  ready  for  use. 

e.  Preparing  for  the  Market. — The  vaccine  is  now  put  into  small  glass 
tubes  and  marketed  as  glycerinated  tube  virus.     The  vaccine  should  be 
kept  in  a  cool,  dry  place.     It  deteriorates  gradually  and  the  time  limit  of 
usefulness  is  stamped  on  each  package. 

The  old  time  ivory  tips  are  still  on  the  market  and  are  preferred  by 
many  physicians.  A  dry  bulk  form  of  the  virus  is  also  marketed.  The 
••ft******"  of  the  use  and  the  action  of  the  virus  are  universally  known.  As 


SEROLOGY — MANUFACTURE    AND    USE    OF    SERA    AND    VACCINES       275 

now  prepared  the  remedy  is  absolutely  safe.  No  ill  effects  ever  follow  its 
use.  Of  the  millions  of  persons  inoculated  within  jecent  years,  there 
probably  has  not  been  a  single  instance  of  bad  effects  which  could  be 
traced  primarily  to  the  vaccine  virus  itself.  A  small-pox  vaccination  is  not 
nearly  as  likely  to  produce  ill  effects  as  the  customary  hand  shake.  In 
fact  the  latter  operation  does  occasionally  spread  an  infection, 

10.  Hydrophobia  or  Rabies  Vaccine 

Pasteur's  hydrophobia  virus  is  obtained  from  the  spinal  cord  of  rabbits, 
inoculated  with  the  virus  from  a  dog  suffering  with  rabies.  The  inocula- 
tion is  made  into  the  dura  mater  of  the  spinal  cord.  The  rabbit  dies  in 
about  two  weeks.  A  second  rabbit  is  inoculated  from  the  first,  which  dies 
even  sooner,  showing  that  the  toxin  gained  in  virulency  in  its  passage 
through  the  first  animal.  This  is  repeated  until  finally  the  animal  dies  in 
six  or  seven  days  after  inoculation.  Beyond  this  the  virulency  of  the 
poison  cannot  be  increased  and  this  constitutes  the  virus  fixe  (fixed  or 
unchanged  virus)  of  Pasteur. 

The  spinal  cord  of  the  rabbit  dead  of  virus  fixe  is  dried  in  a  glass  cylinder 
with  potassium  hydrate.  The  cylinder  is  placed  in  a  cool  dry  place  and 
each  day  small  bits  of  the  cord  are  cut  off  and  placed  in  a  vial  of  glycerin. 
At  the  end  of  fourteen  days  the  virus  is  no  longer  capable  of  producing 
hydrophobia  in  rabbits,  but  the  animal  inoculated  with  it  can  withstand 
the  thirteen  days  virus  (which  was  preserved  in  the  glycerin)  and  so  on 
down  the  scale,  until  finally  the  rabbit  can  withstand  the  virus  fixe  without 
experiencing  serious  effects. 

In  man  it  is  customary  to  begin  the  treatment  for  rabies  (or  suspected 
rabies)  with  the  nine  day  cord  (hypodermic  injections  of  the  cord  emul- 
sions) and  to  give  each  succeeding  day  a  virus  one  day  stronger,  until 
finally  the  virus  fixe  is  injected  without  producing  untoward  symptoms. 
The  individual  thus  treated  is  now  able  to  withstand  the  much  weaker 
virus  from  a  dog  or  other  animal  suffering  from  rabies.  As  the  result  of 
this  mode  of  treatment  the  mortality  rate  from  rabies  is  now  less  than  i 
per  cent.  (Ravenel).  Those  bitten  by  dogs  (or  wolves,  skunks,  cats) 
suffering  from  rabies  or  suspected  of  suffering  from  rabies,  should  cleanse, 
cauterize  and  disinfect  the  wound  at  once,  and  then  immediately  proceed 
to  a  Pasteur  Institute  and  submit  themselves  for  treatment.  The  ear- 
lier, after  infection,  the  treatment  is  begun  the  more  likely  will  the  results 
be  satisfactory.  The  vaccine  is,  however,  now  so  prepared  as  to  make 
home  treatment  possible.  The  graded  doses  of  the  virus  put  up  in 
sterilized  ampules  are  ready  for  immediate  use  by  the  family  physician. 


276  PHARMACEUTICAL  BACTERIOLOGY 

ii.  Phylacogens 

Phylacogens  are  sterile  aqueous  solutions  or  suspensions  of  metabolic 
substances  or  derivatives  generated  by  bacteria  grown  in  artificial  culture 
media.  The  bacteria  are  then  killed  and  filtered  through  clay  filters. 
Each  specific  phylacogen  consists  of  equal  parts  of  the  products  of  the 
infection  and  a  pure  culture  of  the  principle  infecting  organism.  Their 
use  is  based  upon  the  idea  of  Dr.  Schafer  who  holds  the  opinion 
that  most,  if  not  all  infections,  are  mixed,  instead  of  simple,  and  that  the 
organisms  associated  with  the  chief  infecting  organism  play  an  equally 
important  part  in  the  disease  and  in  developing  the  immunization.  A 
number  of  these  preparations  have  been  tried  out  in  practice  but  the  only 
one  which  appears  to  have  met  with  any  considerable  success  is  the  one 
for  rheumatism. 

12.  Mixed  Bacterins.     Polyvalent  Bacterins 

These  have  been  sufficiently  explained  under  sensitized  bacterins. 
It  would  appear  (as  indicated  under  u)  that  certain  infections  are  nor- 
mally multiple.  In  such  cases  it  is  manifestly  unreasonable  to  expect  best 
results  from  a  univalent  bacterin,  made  from  one  of  the  several  infecting 
organisms.  Unless  it  can  be  proven  experimentally  that  one  particular 
organism  in  a  multiple  infection  is  the  primary  cause,  it  is  to  be  assumed 
that  the  best  effects  are  to  be  obtained  from  a  bacterin  made  up  of  all 
of  the  infecting  organisms,  combined  in  about  the  same  proportion  as  they 
occur  in  the  infection.  Such  mixed  bacterins  have  been  tried  out  in_ery- 
sipelas  and  in  cancer,  but  apparently  without  any  considerable  success. 


CHAPTER  XII 

ADENOLOGY.    THE  ENDOCRINOUS  GLANDS  AND  THEIR 

EXTRACTS 

Adtnology  is  the  science  which  treats  of  glands,  their  structure,  func- 
tion and  uses  in  the  animal  economy.  The  functional  activities  of  the 
glands  with  ducts  have  been  known  for  a  long  time,  but  it  is  only  within 
recent  years  that  the  ductless  glands  have  received  serious  attention. 
Sajous  proposed  the  term  hemadenology  for  the  science  which  treats  of  the 
ductless  glands,  which  translated  into  English  means  the  science  of  blood 
glands.  The  German  scientists  spoke  of  the  ductless  gland  as  Blutdriisen 
or  Blutgefassdriisen,  because  of  the  fact  that  these  glands  were  highly 
vascular  and  formed  certain  substances  which  were  poured  into  the  blood 
circulation.  The  term  endocrinology  is  also  much  used  meaning  the  science 
of  internal  secretions,  the  secretions  of  the  ductless  glands  and  also  of  the 
duct  glands  which  pass  into  the  general  circulation  directly,  being  so 
designated.  Since  products  derived  from  glands  with  ducts  are  used 
medicinally,  the  term  hemadenology  is  not  suitable,  and  the  terms 
adenology  and  endocrinology  are  etymologically  more  correctly  applicable. 

The  recent  investigations  in  glandular  functions  have  demonstrated 
that  the  subject  of  immunology  is  intimately  bound  up  with  the  activities 
of  the  glands  and  of  the  body  cells.  It  is  now  known  that  the  duct  glands 
not  only  secrete  substances  which  leave  the  gland  by  way  of  the  duct, 
but  certain  other  illy  denned  though  important  end  products  of  cellular 
activity,  which  pass  directly  into  the  blood  circulation  by  way  of  the 
ultimate  capillaries;  that  is,  these  glands  also  secrete  true  endocrine  sub- 
stances, similar  to  those  secreted  by  the  ductless  glands.  From  this  it 
would  appear  that  the  dividing  line  between  duct  gland  and  ductless  gland 
cannot  any  longer  be  sharply  drawn,  at  least  not  from  the  viewpoint  of 
functional  activities. 

The  study  and  use  of  glands  and  of  glandular  secretions  is  not  by  any 
means  recent.  As  early  as  600  B.C.  testicular  extracts  were  used  in  the 
treatment  of  obesity.  The  keen  interest  in  overcoming  the  excessive 
accumulation  of  adipose  tissue  was  occasioned  by  the  fact  that  those 
good  senators  of  Sparta  and  also  of  Rome,  who  through  a  life  of  indolence 
and  gluttony,  had  become  excessively  obese  were  liable  to  be  haled  before 
a  committee  and  threatened  with  dismissal  from -an  easy  and  lucrative  job 
unless  they  reduced  decidedly  and  forthwith.  The  modern  interest  in 
internal  secretions  dates  back  about  thirty  years,  and  is  clearly  traceable  to 

277 


278  PHARMACEUTICAL  BACTERIOLOGY 

the  earlier  efforts  of  Brown-Sequard.  Brown-Sequard  was  a  keen  investi- 
gator endowed  with  a  fertile  imagination.  He  assumed  that  all  tissues 
gave  off  or  secreted  substances  to  the  blood  which  were  essential  to  life 
and  which  were  peculiar  to  each  kind  of  animal.  He  made  the  mistake 
of  taking  the  public  into  his  experimental  confidence  with  the  result  that 
his  work  was  made  so  ridiculous  through  the  lay  press  that  he  became 
entirely  discouraged  and  all  that  he  did  was  discredited  and  soon  almost 
entirely  forgotten,  to  be  again  revived  within  two  decades. 

Until  about  thirty  years  ago,  the  physiologists  gave  practically  no 
attention  to  the  ductless  glands,  merely  mentioning  them  and  suggesting 
that  they  were  functionless  vestigial  remnants  of  once  larger  and  iunction- 
ally  active  glands.  This  idea  based  on  ignorance  had  the  effect  of  en- 
couraging surgical  removal  of  the  supposedly  useless  structures,  for  little 
or  no  cause.  Today,  for  example,  the  removal  of  the  tonsils  has  become  a 
craze,  equalled  only  by  the  wholesale  extraction  of  teeth  and  the  snipping 
of  vermiform  appendices.  Dr.  Williams  (in  the  Practitioner,  Jan.  1915) 
makes  this  terse  and  truthful  statement  which  is  equally  applicable  to 
the  operative  procedures  above  indicated.  "The  truth  is,  these  operative 
procedures  (in  Grave's  disease)  represent  the  heroic  application  of  loose 
conclusions  from  insufficient  data." 

The  work  done  on  internal  secretions  since  1889  has  demonstrated 
the  following: 

1.  All  of  the  glands,  those  with  ducts  as  well  as  those  without  ducts, 
secrete  substances  which  are  thrown  back  into  the  circulation  and  which 
are  more  or  less  essential  to  the  normal  functioning  of  the  body. 

2.  Some  of  the  glandular  secretions  are  absolutely  essential  to  life, 
as  those  of  the  suprarenals,  the  parathyroids  and  the  pancreas;  while 
others  are  not  essential  to  continued  life,  as  those  of  the  testes,  the  ovaries, 
the  spleen  and  the  tonsils. 

3.  The  functional  activities  of  most  of  the  glands,  if  not  all  of  them,  is 
interrelated,  and  again  the  activities  of  all  of  the  glands  are  interrelated 
with  the  functional  activities  of  all  of  the  somatic  body  cells  and  with  the 
germatic  cells.     That  is,  any  serious  disturbance  of  any  one  gland  is  apt 
to  react  upon  the  activities  of  the  other  glands  and  upon  the  body  cells. 
On  the  other  hand,  the  disturbance  of  the  function  of  the  body  cells  will 
react  upon  the  activities  of  the  glands. 

4.  A  lessening  of  a  glandular  function,  or  of  the  functioning  of  body 
cells  is  apt  to  be  compensated  by  an  increased  or  otherwise  modified 
functioning  of  some  other  gland  or  glands.    Law  of  compensating  bodily 
functions. 

The  glands  in  general  secrete  substances  which  influence  the  activities 
of  the  bodily  organs.  Chemical  substances  of  this  kind,  which  stimulate 


ADENOLOGY.   THE  ENDOCRINOUS  GLANDS  AND  THEIR  EXTRACTS   279 

the  functional  activities  of  organs  are  called  hormones.  Shafer  called 
attention  to  the  fact  that  apparently  some  glandular  products  inhibited 
or  checked  or  retarded  the  normal  functional  activities  of  organs"  to 
which  the  name  chalones  has  been  applied.  The  chalones  may  be  com- 
pared to  a  balance  wheel,  tending  to  regulate  the  normal  growth  and 
functioning  of  tissues  and  organs.  Without  the  chalones  there  would  be 
hyper-function  due  to  the  unchecked  hormones. 

The  function  of  a  gland  may  be  excessive,  or  lessened,  or  abnormal 
being  neither  excessive  nor  subnormal.  Thus  we  have  a  hyper-  a  hypo- 
and  a  dys-function  of  a  gland  or  of  glands.  Recent  investigations  and 
observation  have  shown  that  the  dys-function,  to  a  lesser  degree  also 
the  hyper-  and  hypo-function  of  glands,  modify  profoundly  the  resistance 
to  infections.  In  other  words,  the  glands  are  of  the  greatest  importance 
form  the  viewpoint  of  immunology.  This  makes  it  clear  why  the  glandular 
extracts  and  secretions  have  recently  come  into  use  in  medical  practice. 
The  results  of  their  use  have  in  some  instances  been  marvelous,  whereas 
in  other  instances  the  therapeutic  effects  have  been  almost  nil.  It  may 
be  stated,  however,  that  the  therapeutic  application  of  glandular  extracts 
is  as  yet  in  its  infancy  and  is  based  almost  entirely  upon  empiricism.  The 
following  is  a  brief  review  and  summary  of  the  science  of  adenology. 

i.  The  Glands  with  Ducts 

1 .  The  Liver. — This  is  the  largest  gland  of  the  body  and  it  is  essential 
to  life.     The  glycogenic  function  of  this  gland  is  familiar  to  students  of 
physiology.     It  gives  off  two  substances  within  the  cells,  namely  glycogen 
and  urea,  which  are  poured  into  the  blood  for  the  purpose  of  general 
nutrition  or  for  elimination.     Gay  has  recently  isolated  a  substance 
from  animal  livers  and  also  found  abundantly  in  certain  mussels   (the 
abalone  of  the  Pacific  Coast),  to  which  he  has  given  the  name  "taurin" 
and  which  promises  to  be  a  curative  agent  in  tuberculosis.     The  liver  is 
one  of  the  organs  which  is  usually  quite  free  from  tuberculosis  and  the 
supposition  is  that  it  contains  a  substance  (taurin)  which  prevents  tuber- 
cular infection.     The  laboratory  experiments  on  tubercular  guinea  pigs 
have  been  very  promising. 

2.  The  Spleen. — Apparently  the  spleen  is  not  essential  to  life  as  has 
been  shown  by  animal  experiments.     The  chief  changes  following  extir- 
pation are  enlargement  of  the  lymphatic  glands  and  a  hyper-function  of  the 
red  marrow  of  the  long  bones.    The  spleen  has  been  credited  with  giving 
rise  to  leucocytes  and  is  supposed  to  be  the  grave  yard  of  the  red  blood 
corpuscles.     The  true  function  of  the  spleen  is  not  yet  known. 

3.  The  Pancreas. — The  pancreas  is  absolutely  essential  to  life,  as  its 
removal  results  in  death,  preceded  by  a  pronounced  glycosuria  or  diabetes 


280  PHARMACEUTICAL  BACTERIOLOGY 

mellitus,  with  the  accompanying  symptoms  of  polyuria,  great  thirst  and 
hunger  and  some  acidosis.  The  experimental  evidence  indicates  that  the 
pancreas,  in  addition  to  its  usual  function  in  digestion,  secretes  a  substance 
which  is  essential  to  the  normal  bodily  metabolism  of  sugar,  which  sub- 
stances (secretins)  are  probably  formed  in  the  so-called  island  of 
Langerhans. 

The  following  statements  pertaining  to  the  endocrine  secretions  of 
the  testes,  the  ovaries  and  the  mammary  glands  are  taken  from  a  series 
of  popular  lectures  on  biological  products  by  Parke,  Davis  and  Company. 

4.  The  Testes. — The  part  played  by  the  testes  as  internal  secreting 
organs  is  inversely  shown  by  what  takes  place  after  castration.     The 
castrated  rooster  experiences  a  shriveling  ofthe  comb,  wattles,  and  spurs, 
the  character   of  the  voice  is  changed,  the  neck  and  tail  feathers  are 
poorly  developed,  and  there  is  an  excessive  deposit  of  fat  and  he  becomes 
more  docile. 

Castration  of  food-producing  animals,  especially  cattle,  is  often  per- 
formed because  of  the  increased  tendency  toward  fat  deposits,  and  the 
change  in  the  consistency  of  the  muscular  tissues  produced,  this  rendering 
such  tissues  more  suitable  for  food;  and  the  operation  makes  the  animal 
more  docile  and  quiet. 

The  best  opportunity  of  determining  the  effect  of  castration  on  man 
has  been  afforded  by  the  custom  in  certain  Eastern  countries  of  thus 
muttilating  harem  guards.  This  practice  is  also  resorted  to  to  some 
extent  by  a  religious  sect  in  Russia,  the  "Skopzen;"  and  in  Italy  it  was 
formerly  not  an  uncommon  procedure  to  castrate  male  singers  during  child- 
hood in  order  that  they  might  retain  the  juvenile  tone  and  fibre  of  the  voice. 
If  the  operation  is  performed  in  early  life  it  results  in  an  absence  of  sexual 
power  and  in  infantile  development  of  the  external  genitals.  Castrates 
do  not  possess  the  courage,  passions  and  aspirations  of  normal  men,  and 
they  appear  to  be  lacking  in  the  higher  artistic  endowments.  They  are 
said  to  be  tricky,  revengeful,  and  cruel.  Their  intellectual  abilities  are 
not  impaired  to  any  considerable  extent,  as  many  eunuchs  have  been 
men  of  more  than  the  average  intelligence. 

While  the  existence  of  an  internal  secretion  of  the  testes  is  definitely 
established  and  its  far-reaching  importance  clearly  recognized,  the  use  of 
testicular  preparations  in  therapeutics  has  never  achieved  any  great 
degree  of  success!  Many  years  ago  the  first  attempts  to  apply  products 
of  this  kind  were  made,  and  it  was  claimed  that  such  treatment  brought 
about  an  increase  in  physical  and  mental  vigor.  Subsequent  investiga- 
tions have  not  made  this  claim  good. 

5.  The  Ovaries. — We  have  abundant  evidence  of  the  importance  of  the 
ovary  as  an  internal  secreting  gland.     We  know,  for  instance,  that  there 


ADENOLOGY.      THE  ENDOCRINOUS  GLANDS  AND  THEIR  EXTRACTS      281 

is  an  intimate  relationship  between  the  ovary  and  the  menstrual  function. 
When  the  ovaries  are  completely  removed,  menses  are  terminated.  Pre- 
sumably the  institution  of  menses  is  due  to  some  change  in  the  glandular 
function  of  the  ovary  at  puberty,  and  it  is  noteworthy  that  at  this  time 
also  the  development  of  the  distinctively  feminine  characteristics  of  form 
and  feature  take  place,  these  being  stimulated,  it  is  thought,  by  the  ovarian 
secretion. 

The  changes  which  occur  at  the  menopause,  both  in  the  physical  and 
mental  characteristics  of  the  woman,  suggest  that  here,  too,  we  have  a 
marked  alteration  in  the  character  of  the  ovarian  activity;  apparently 
there  is  a  cessation  of  that  phase  of  the  function  which  is  instituted  at 
puberty. 

It  was  formerly  believed  that  the  internal  secretion  of  the  ovary  was 
elaborated  by  the  corpus  luteum.  The  corpus  luteum  in  the  non-preg- 
nant state  degenerated  and  shrinks  up  in  a  very  short  time,  but  if  the 
ovum  has  been  impregnated  it  persists  for  several  months.  It  is  now 
known  that  the  corpus  luteum  is  not  the  sole  source  of  the  secretion  of  the 
ovary,  and  by  many  it  is  believed  that  it  is  not  even  the  more  important 
one.  One  thing  is  certain,  however:  the  corpus  luteum  does  represent 
an  active  element  of  internal  secretion. 

Both  corpora  lutea  and  desiccated  ovarian  glands  (ovarian  substance) 
have  been  extensively  used  in  the  treatment  of  natural  as  well  as  arti- 
ficial "change  of  life."  The  best  results  have  been  obtained  in  the  treat- 
ment of  artificial  menopause — by  " artificial"  meaning  cases  in  which  for 
some  reason  it  has  been  necessary  to  remove  the  ovaries  completely  or 
in  part.  In  other  words,  these  products  are  of  advantage  in  cases  where 
an  insufficient  amount  of  ovarian  tissue  is  present  to  adequately  supply 
the  physiological  demands  of  the  body.  The  symptoms  commonly  oc- 
curring are  often  completely  controlled. 

Ovarian  treatment  has  been  used  with  some  success  in  infantilism  of  the 
genital  organs,  but  is  must  be  borne  in  mind  that  such  conditions  are  often 
associated  with  pituitary  disease.  The  vomiting  of  pregnancy  is  occa- 
sionally relieved  by  ovarian  treatment,  too,  but  as  a  rule  this  condition 
responds  better  to  the  use  of  suprarenal  extracts. 

6.  The  Mammary  Gland. — It  has  not  been  definitely  established  that 
the  mammary  glands  have  a  function  other  than  the  secretion  of  milk, 
but  there  is  considerable  reason  to  believe  that  they  elaborate  some  sub- 
stance which  influences  ovarian  activity;  certainly  there  is  no  question 
as  to  the  intimate  relationship  existing  between  the  ovaries  and  mamma- 
ries.  It  is  interesting  to  note,  however,  that  the  striking  development 
of  these  glands  during  pregnancy  is  quite  independent  of  the  ovaries; 
if  the  ovaries  be  completely  removed  from  pregnant  animals,  the  mamma- 


282  PHARMACEUTICAL  BACTERIOLOGY 

ries  develop  just  the  same  and  lactation  is  not  interfered  with.  The  same 
v  phenomenon  has  been  observed  in  women  from  whom  for  some  reason 
or  other  it  has  been  necessary  to  remove  the  ovaries  during  the  gestation 
period. 

It  has  been  claimed  by  some  investigators  that  the  development  of 
the  mammaries  during  pregnancy  is  dependent  upon  secretory  activity  of 
the  placenta  ("after-birth").  They  have  urged  in  support  of  this  theory 
that  mammary  development  persists  in  pregnant  animals,  regardless 
of  the  death  of  the  fetus,  just  as  long  as  the  placenta  remains.  Further- 
more, placental  extracts  have  been  reported  to  be  active  stimulants  to 
lactation.  Other  evidence  seems  to  support  the  assumption  that  there  is 
some  substance  derived  from  the  fetus  which  stimulates  the  mammaries, 
and  the  injection  of  fetal  extracts  in  normal  animals  brings  about  an  en- 
largement of  the  mammary  glands.  Possibly  both  factors  are  involved. 

Therapeutically,  the  importance  of  a  product  made  from  the  mammary 
gland  seems  to  lie  in  its  effect  in  neutralizing  excess  ovarian  secretion,  and 
this  has  resulted  in  a  preparation  of  this  character  being  applied  to  the 
control  of  menstrual  excesses  having  their  origin  in  over-functionating  of 
the  ovaries.  A  still  more  interesting  application  of  mammary  gland  treat- 
ment has  been  in  fibroid  tumors  of  the  uterus.  On  first  consideration 
such  a  therapeutic  measure  looks  preposterous,  but  the  theory  is  that 
fibroids  have  their  origin  to  a  large  extent  in  uterine  congestion.  Sev- 
eral investigators  have  reported  that  the  use  of  mammary  extracts  has 
resulted  in  an  arresting  or  disappearance  of  these  fibroids,  and  while  the 
evidence  thus  far  produced  is  by  no  means  conclusive,  the  possibilities 
of  such  treatment  warrant  further  study. 

2.  The  Ductless  Glands 

The  ductless  glands  are  without  ducts,  they  are  small  in  comparison 
with  the  duct  glands  and  occupy  well  protected  positions  in  the  body. 
They  consist  mainly  of  epithelial  cells  which  are  in  close  relation  to  the 
walls  of  capillary  blood  vessels  and  lymphatics,  and  in  some  instances,  if 
not  all,  under  the  control  of  the  cerebro-spinal  nerve  system.  By  reason 
of  their  relationship  to  the  blood  circulation,  they  have  been  called  vas- 
cular glands  and  blood  glands.  Some  recent  observations  would  indicate 
that  the  sympathetic  nerve  supply  has  a  very  intimate  relationship  to 
the  ductless  glands  as  well  as  to  the  epithelial  cellular  structure  of  the 
capillaries. 

While  there  is  much  which  is  as  yet  undetermined  with  regard  to  the 
ductless  glands,  there  is  much  that  can  be  said  with  some  degree  of  definite- 
ness.  They  most  certainly  secrete  substances  which  are  necessary  to 
the  proper  or  normal  correlative  functioning  of  organs.  They  contain 


ADENOLOGY.   THE  ENDOCRINOUS  GLANDS  AND  THEIR  EXTRACTS   283 

protective  and  defensive  measures  against  disease.  They  regulate  .the 
function  of  ovulation,  of  sex  development,  of  pregnancy,  of  muscular 
tonicity,  of  vascular  tonicity,  of  adiposity,  the  growth  of  tissues,  sugar 
metabolism,  glandular  activity,  etc.,  etc.  Although  the  glands  are  far 
apart  in  the  body,  there  is  nevertheless  a  functional  intercommunication 
between  them.  They  have  furthermore  a  compensatory  interaction,  an 
altered  function  in  one  gland  being  balanced  by  the  functional  activities  of 
one  or  more  other  glands.  Again,  a  serious  dys-function  of  one  gland  may 
upset  the  functional  activities  of  the  others. 

Every  cell  of  the  body  may  be  likened  to  a  ductless  gland  capable  of 
taking  on  some  one  or  other  of  the  functional  activities  of  the  glands,  and 
all  of  the  functionally  active  body  cells  do  cooperate  with  the  glands  in  the 
maintenance  of  the  bodily  functions.  The  significance  and  importance  of 
the  intelligent  use  of  ductless  gland  products  are  clearly  set  forth  by  Sajous 
as  follows. 

"What  are  termed  'backward  childern'  aggregate,  judging  from  the 
proportion  shown  by  the  public  schools  of  Philadelphia,  318,000  in  the 
public  schools  of  the  United  States.  As  this  does  not  include  children  who 
are  too  young  to  attend  school,  an  estimate  of  one  million  of  backward 
children  of  all  ages  in  the  whole  country,  would  be  nearer  the  true  figure. 
These  children,  usually  deemed  merely  deficient  in  capacity  of  spontaneous 
attention  and  memory  and  believed  to  show  no  evidences  of  degeneracy, 
possess  in  many  instances  stigmata  which  point  directly  to  impairment, 
through  heredity  or  local  lesions,  of  the  ductless  glands,  and  due  in  many 
instances  to  one  or  more  "  children's  diseases."  We  may  witness  one  or 
more  signs  of  hypothyroidism  or  larval  myxedema,  with  mental  torpor, 
hypothermia,  and  perhaps  a  little  pallor  as  only  signs;  or  close  examination 
may  elicit  a  mild  form  of  cretinism,  with  slightly  stunted  growth,  a  pug 
nose,  thick  lips,  a  somewhat  harsh  skin — children  who  often  show  decayed 
teeth  and  a  predilection  for  tonsillitis. 

A  lower  grade  still  of  these  (often  redeemable)  degenerates  show  defects 
of  speech  and  ideation  and  deficient  capacity  of  spontaneous  attention. 
Left  untreated,  such  subjects  usually  drift  to  the  category  of  "idiots," 
a  blind  devotion  to  tradition  having  associated  these  unfortunates  with 
"heredity" — a  fit  companion  for  "idiosyncrasy"  as  a  cloak  for  ignorance. 
Hemadenology  will  do  much  to  tear  asunder  the  clouds  which  hover  over 
this  great  question.  Indeed,  through  the  efforts  of  eugenists  to  protect 
future  generations,  the  unfortunates  of  our  own  generation  are  increasingly 
exposed  to  injustice.  This  will  cease  when  the  prevailing  tendency  to 
overlook  the  flood  of  light  which  modern  contributions  to  our  knowledge 
concerning  the  ductless  glands  have  thrown  upon  heredity  will  inspire  the 
labors  of  these  well  meaning  scientists. 


284  PHARMACEUTICAL  BACTERIOLOGY 

In  contrast  with  these  defectives  are  the  cases  of  infantiUsm,  charac- 
terized by  the  persistence  of  the  physical  and  mental  characteristics  of 
childhood,  but  without  idiocy  or  dwarfism.  The  miniature  men  of  the 
Lorain  type  and  the  defectives  of  the  Mongolian  type  with  their  slanting 
eyes,  bulging  foreheads,  are  examples  of  this  class,  due  in  most  instances 
to  defects  in  the  upbuilding  of  the  organism,  a  process  in  which  all  the 
ductless  glands  take  part.  Still  another,  though  rarer,  type  is  the  infan- 
tilism of  pituitary  origin — obesity  with  feminism  in  the  distribution  of  fat, 
the  nates,  thighs,  and  breasts  especially,  but  with  deficient  development  of 
the  sexual  organs,  a  moon  face,  and  weak  mentality. 

Crime  presents  aspects  which  also  belong  to  the  field  of  the  hemadeno- 
logist.  All  the  types  described  above  are  usually  docile,  the  exceptions 
being  some  cases  of  the  pituitary  adiposogenital  type,  and  show  but  little 
if  any  predilection  for  vice.  Among  those  recorded  as  imbeciles,  who  show 
deficient  intelligence  and  loquaciousness,  abnormally  good  memory, 
untruthfulness,  arrogance,  and  maliciousness,  may  sometimes  be  dis- 
cerned types,  which  owing  to  the  landmarks  of  defective  development 
resulting  from  imperfect  balance  of  ductless  gland  activities,  point  to 
links  between  the  latter  and  crime.  To  seek  these  associations,  restore 
normal  equipoise  in  the  production  of  hormones,  thus  insuring  normal 
metabolism  in  all  tissues,  particularly  the  osseous  and  central  nervous 
systems  when  it  is  still  time,  offers  broad  avenues  of  hope  for  the  redemp- 
tion of  some  of  these  unfortunates  from  the  drifts  of  iniquity. 

'Insanity  likewise  claims  the  attention  of  the  hemadenologist.  The 
psychoses  of  exophthalmic  goitre  and  myxedema,  and  the  idiocy  of  micro- 
cephaly due  to  inadequacy  or  absence  of  the  adrenals,  are  familiar  exam- 
ples. Dementia  praecox,  which  is  stated  to  initiate  twenty-five  per  cent, 
of  the  cases  of  insanity  harbored  in  our  asylums,  is  increasingly  being 
shown  to  be  closely  related  to  perverted  action  of  the  same  glands,  various 
types  of  the  disease  being  represented  by  a  corresponding  number  of  forms 
of  abnormal  glandular  action.  Such  being  the  case,  we  are  brought  to 
realize  the  many  directions  in  which  abnormal  activity  of  the  ductless 
glands  may  affect  mentality.  Beside  the  enfeeblement  of  the  mind 
characterized  by  unequal  weakening  of  the  faculties,  emotion,  judgment, 
self  control,-  etc.,  we  witness  impulsive  actions,  flightiness,  catalepsy, 
automatic  obedience,  vergiberation,  mutism,  delusions,  hallucinations, 
etc.  The  field  is  thus  prolific  in  its  opportunities  for  the  elucidation  of 
many  of  the  complex  problems  with  which  psychiatrists  are  confronted  in 
respect  to  the  genesis  of  psychoses. 

Obesity  in  all  its  forms  normally  falls  within  the  scope  of  hemadenology. 
Beside  the  familiar  varieties  due  to  overuse  of  carbohydrates,  defective 
oxidation,  etc.,  there  are  types  which,  as  is  well  known,  are  due  to  defi- 


ADENOLOGY.   THE  ENDOCRINOUS  GLANDS  AND  THEIR  EXTRACTS   285 

cient  thyroid  activity,  which  entails  from  my  viewpoint,  impaired  ac- 
tivity of  all  other  ductless  glands.  In  children  we  may  have  also  the 
adipositas  cerebralis  of  Frohlich,  in  which  general  obesity  occurs  with 
defective  development  of  the  sexual  organs  and  impaired  intelligence — 
due  to  deficient  pituitary  activity.  Closely  allied  genetically  with  these 
cases,  are  those  showing  the  adiposogenital  syndrome  of  Launois,  very 
similar  to  Frohlich's,  but  without  impairment  of  intelligence.  The  adipo- 
sis  dolorosa  of  Dercum,  in  which  there  is  obesity,  general  or  localized  in 
areas,  with  pain,  spontaneous  or  paroxysmal,  is  also  ascribed  to  impaired 
activity  of  certain  ductless  glands.  Still  another  type,  symmetrical  lipo- 
matosis,  is  characterized  by  the  presence  of  masses  of  fat,  often  tender  or 
the  seat  of  spontaneous  pain,  symmetrically  in  the  axillae,  groin,  or  other 
regions,  but  oftenest  about  the  neck.  Finally,  the  obesity  of  pineal 
deficiency  may  be  mentioned  as  another  example  of  the  close  relationship 
between  the  ductless  glands  and  obesity. 

Falling  to  the  lot  of  the  hemadenologist  also  are  the  abnormalities  of 
growth,  several  of  which,  even  in  individuals  in  apparent  health,  are  mani- 
festations, active,  latent,  or  extinct,  of  some  morbid  process.  In  acro- 
megaly  for  example,  we  may  have  general  enlargement  of  the  body,  espe- 
cially of  the  extremities  and  face;  the  lips,  nose,  and  chin  are  more  or 
less  prominent  and  there  is  general  increase  of  massiveness  of  the  frame. 
Individuals  presenting  such  a  type  are  not  uncommon;  in  these,  as  well 
as  in  certain  very  tall  subjects,  temporary  lesions  of  the  pituitary, 
awakened  by  some  acute  febrile  process,  may  have  caused  the  acrome- 
galic  process  to  proceed  far  enough  to  provoke  the  appearance  of  its  most 
salient  phenomena — all  incapable  of  retrogression,  after  the  causative 
morbid  process  in  the  pituitary  proper  has  disappeared. 

Resembling  such  cases  at  times  are  those  of  adrenal  tumor,  some  of 
which  cause  premature  development  so  marked  in  rare  instances  that  a 
child  of  eight  years  may  attain  the  size  of  an  adult.  The  adipositas  cere- 
bralis of  Frohlich  and  the  adiposogenital  syndrome  of  Launois  also  sug- 
gestive of  acromegaly  in  some  cases,  are  deemed  extremely  rare  because 
the  fully  developed  morbid  process  is  alone  taken  as  standard.  Here  and 
there,  however,  the  trained  eye  of  the  hemadenologist  may  discern  the 
stigmata  of  these  disorders,  and  oppose,  through  compensative,  regulative 
or  inhibitive  measures,  their  evil  trend. 

Stunted  growth  as  clearly  belongs  to  the  do  main  of  the  hemadenologist. 
This  may  follow,  also  irrespective  of  any  other  abnormal  effect,  the  infec- 
tions of  childhood,  especially  where  the  thyroid,  thymus,  and  adrenals 
had  been  the  seat  of  lesions.  In  the  complicated  types  there  is,  beside 
the  dwarfism  of  cretinism  and  its  congerers,  Mongolian  and  Loraine 
infantilism,  the  victim  of  achondroplasia,  of  fetal  rickets  whose  lifelong 


286  PHARMACEUTICAL  BACTERIOLOGY 

mortification  is  intensified  by  the  fact  that  unlike  that  of  the  other  types, 
his  mind  is  as  alert  as  that  of  the  normal  individual.  His  large  head,  saddle 
nose,  short  and  bowed  legs,  prominent  abdomen,  and  marked  lordosis, 
bespeak  little  indeed  in  favor  of  a  medical  science  which  cannot  check  the 
development  of  such  deformities  in  their  incipiency. 

Myxedema,  cretinism,  and  other  classic  disorders  of  the  various  duct- 
less glands  obviously  belong  to  the  field  of  the  hemadenologist.  It  should 
be  borne  in  mind,  however,  that  in  their  larval  or  mild  forms  they  con- 
stitute in  many  instances,  the  so-called  rebellious  cases  met  with  in  general 
practice.  The  sufferer  of  larval  hypothyroidism,  for  example,  may  show 
little  else  than  occipital  or  interscapular  pain  and  cold  extremities  and  yet 
resist  all  the  antirheumatic  or  antineuralgic  measures  that  a  century  may 
have  suggested.  Unrecognized,  such  patients  sometimes  contribute  to 
their  physician's  diagnostic  acumen  by  becoming  frank  cases  of  myxe- 
dema — when  organotherapy  arrest  both  the  latter  and  the  rheumatism. 
Lying  behind  tetany,  paralysis  agitans,  and  osseous  disorders  are,  it  is 
believed,  lesions  of  the  parathyroid  glands — which  thus  become,  as  does  the 
thyroid,  elucidative  factors  in  obscure  though  relatively  commonplace 
disorders. 

Much  the  same  remarks  apply  to  larval  Addison's  disease.  While 
more  or  less  bronzing  characterizes  the  latter,  we  often  meet  in  pale  children, 
neurasthenic  adults,  and  premature  seniles,  the  typical  signs  of  this  con- 
dition, asthenia,  sensitiveness  to  cold,  cold  extremities,  hypotension,  weak 
cardiac  action  and  pulse,  anorexia,  anemia,  constipation,  etc.,  but  without 
bronzing.  Acute  febrile  diseases,  pneumonia,  diphtheria,  typhoid  fever, 
etc.,  may  bring  on  a  similar  state  by  exhausting  the  adrenals,  the  patient 
dying  after  a  period  of  weak  heart,  low  blood  pressure,  asthenia,  a  tend 
ency  to  fainting,  prostration,  etc., — an  issue  which  a  few  timely  doses  of 
adrenaline  in  saline  solution  would  have  prevented.  Excessive  activity 
of  the  adrenals  is  another  cause  of  death  in  children  seldom  recognized. 
Here  the  work  of  the  hemadenologist  will  become  elucidative  and  life 
saving. 

Goitre  and  exophthalmic  goitre,  the  bulk,  as  it  were,  of  the  cases 
witnessed  by  the  hemadenologist,  need  his  special  intervention,  to  elimi- 
nate at  the  earliest  moment,  that  of  the  surgeon.  While  in  no  way  dis- 
crediting the  value  of  operative  procedures  in  appropriate  cases,  my  own 
experience  confirms  that  of  Leonard  Williams  in  condemning  promiscuous 
resort  to  the  knife.  This  applies  also  to  many  cases  of  ordinary  goitre. 
Many  patients  subjected  to  operation  could  have  been  cured  by  medical 
treatment,  thus  preserving  for  them  a  useful  organ.  We  must  not  lose 
sight  of  the  fact,  however,  that  much  work  remains  to  be  done  to  establish 
the  precise  limitations  between  operable  and  inoperable  cases — a  line 


ADENOLOGY.   THE  ENDOCRINOUS  GLANDS  AND  THEIR  EXTRACTS   287 

or  research  which  the  hemadenologist  should  carry  to  an  early 
termination. 

The  thymus  in  various  ways  claims  the  attention  of  the  hemadeno- 
logist. Its  temporary  existence  associated  it  with  development  and  par- 
ticularly with  that  of  the  brain  and  osseous  system.  Idiocy,  thymic  as- 
phyxia, and  status  lymphaticus  are  doubtless  but  a  few  of  the  disorders 
the  thymus  may  awaken.  Adenoids,  enlarged  tonsils,  and  rickets  are 
kindred  conditions  which  greater  knowledge  concerning  the  functions  of  the 
thymus  will  tend  great*  y  to  elucidate.  Rickets  and  stunted  growth 
belong  to  the  same  category. 

The  reproductive  organs  present  features  which  distinctly  belong  to 
the  domain  of  the  hemadenologist.  What  knowledge  of  the  functions  of 
the  internal  secretions  of  the  testes,  ovaries,  and  corpora  lutea  has  already 
been  garnered,  has  contributed  much  to  our  therapeutic  resources  in 
conditions  which  formerly  found  us  relatively  powerless.  The  observa- 
tions of  Brown-Sequard  to  the  effect  that  the  energy  of  the  nerve  centres 
and  cord  is  stimulated  and  that  the  individual  is  endowed  with  physical, 
moral,  and  intellectual  characteristics  of  sex  by  means  of  orchitic  injec- 
tions, denote  a  wide  field  of  usefulness,  beside  the  treatment  of  sexual 
impotence.  Menopause,  physiological  and  post-operative,  amenorrhea, 
and  kindred  disorders  of  the  female  studied  adequately  from  this  new  view- 
point cannot  but  prove  fruitful." 

The  following  is  a  brief  summary  of  the  functional  activities  and  uses 
of  the  ductless  glands. 

i.  The  Tonsils. — These  are  two  almond  shaped  bodies  lying  one  on 
either  side  at  the  entrance  to  the  fauces.  They  are  most  prominent  in 
childhood,  beginning  to  reduce  after  puberty.  These  glands  have  been 
utterly  neglected  by  physiologists  and  practically  nothing  is  known  re- 
garding their  function,  either  actual  or  problematical.  Though  neglected 
by  the  physiologists  and  merely  located  in  space  by  the  anatomists,  they 
have  not  been  neglected  by  the  surgeon.  The  rule  seems  to  be  to  remove 
every  tonsil  that  can  be  removed,  whether  there  is  reason  for  so  doing  or 
not,  and  this  despite  the  fact  that  many  after  effects  of  operations  on 
childern  have  been  most  serious.  The  surgeon  tries  to  explain  these  cases 
by  stating  that  it  was  the  fault  of  the  operation,  the  tonsil  being  only 
partially  removed,  leaving  a  decayed  and  infected  root,  others  that  the 
ill  consequences  should  not  be  laid  to  the  operation,  but  rather  to  the 
delay  in  having  the  tonsils  removed.  In  many  directions  there  is  manifest 
a  tendency  to  some  hesitation  about  removing  tonsils  that  are  sound  or 
that  are  only  slightly  infected.  Various  suggestions  have  been  offered 
as  to  the  function  of  the  tonsils,  all  of  which  are  largely  guessing.  One  is 
that  they  protect  against  infections  to  both  the  respiratory  tract  and  the 


288  PHARMACEUTICAL  BACTERIOLOGY 

digestive  tract.  Another  is  that  they  retard  the  sex  development  of  the 
child,  for  which  assumption  there  appears  to  be  some  justification  in  the 
fact  that  sexually  precocious  children  frequently  have  small  or  rudimentary 
tonsils.  This  suggestion  appears  to  be  negatived  by  the  fact  that  the 
removal  of  the  tonsils  in  young  children  does  not  hasten  or  increase  the 
sex  development  or  give  rise  to  any  signs  of  sexual  precocity. 

2.  The  Pineal  Gland. — Also  known  as  pineal  body  and  epiphysis  cere- 
bri,  was  supposed  to  be  the  vestigial  remnant  of  the  Cyclopean  eye. 
This  is  a  very  small  pine  cone  like  body  projecting  from  the  third  ventricle. 
In  early  life  it  has  a  glandular  structure  and  reaches  its  greatest  develop- 
ment at  about  the  seventh  year.     After  this  period  and  more  especially 
after  puberty  it  loses  its  glandular  appearance  and  gradually  dwindles, 
degenerating  into  a  fibrous  tissue.     In  hypo-function  of  this  gland  and  also 
in  dysfunction,  in  children,  there  is  accelerated  development  of  the  repro- 
ductive organs  with  attendant  mental  precocity.     The  inference  is  there- 
fore, that  the  gland  secretes  a  substance  (a  chalone)  which  inhibits  growth 
and  more  especially  restrains  the  development  of  the  reproductive  glands. 
Total  extirpation  of  the  gland  is  not  fatal. 

3.  The  Pituitary  Body  or  Gland. — Lies  in  the  sella  turcica  of  the  sphenoid 
bone  and  is  usually  described  as  consisting  of  two  parts,  the  larger  anterior 
lobe  of  distinctly  glandular  structure,  and  a  much  smaller  posterior  lobe 
of  nervous  origin  and  composed  of  neuroglia  cells  and  fibers.     Total 
extirpation  of  this  gland,  or  of  its  anterior  lobe  alone,  results  in  death, 
preceded  by  lowering  of  blood  pressure,  of  temperature,  feeble  and  slow 
respiration,  unsteadiness  of  movement,  muscular  twitching,  lethargy  and 
coma.     Occasionally  there  is  also  glycosuria.     Removal  of  the  posterior 
lobe  alone  was  without  marked  effects.     The  secretions  of  the  anterior 
lobe  stimulate  the  growth  of  the  skeleton  and  associated  tissues.     Hyper- 
function  (hyperpituitarism)  in  children  gives  rise  to  giantism,  and  in 
adult  life  to  that  special  growth  of  long  bones  designated  by  acromegaly. 
A  deficiency  of  secretion  gives  rise  to  infantilism,  to  excessive  fatty  tissue 
formation   (adiposis  dolor  osa],  sexual  inactivity  with  actual  atrophy  of 
the  sex  organs,  loss  of  hair,  disturbances  of  nutrition,  reduced  mental 
activity,  etc.     The  hyper-pituitarism  of  adults  manifests  itself  by  the 
elongation  of  the  long  bones  of  the  extremities,  the  hands  and  feet,  in- 
creased angularity  of  the  skeletal  structure,  the  tongue  is  thickened  causing 
a  thick  speech,  mentality  is  lessened;  with  a  characteristic  curvature  of 
the  spinal  column  (cervico-dorsal  kyphosis  with  a  compensatory  lumbar 
lordosis),  there  is  thickening  of  the  skin,  the  chin  projects  and  the  lower 
teeth  generally  project  beyond  the  plane  of  the  upper  teeth;  there  is 
increased  thirst  and  occasionally  glycosuria. 

4.  The    Thymus   Gland. — This    gland    is   largest    during    embryonal 


ADENOLOGY.   THE  ENDOCRINOUS  GLANDS  AND  THEIR  EXTRACTS   289 

development  and  begins  to  dwindle  at  the  end  of  the  second  year,  having 
almost  entirely  disappeared  at  the  age  of  puberty.  It  lies  in  the  neck-on 
the  front  and  sides  of  the  trachea,  consisting  of  two  lobes.  The  chief 
function  of  this  gland  appears  to  be  the  stimulation  of  embryonal  and 
early  infantile  development.  Occasionally  this  gland  persists  during 
adult  life,  in  which  cases  it  is  accompanied  by  excessive  lymphoid  develop- 
ment, known  as  myxcedema.  Thymus  gland  extract  has  been  used 
empirically  in  the  treatment  of  hyperthyroidism,  in  hemophilia,  in  rickets, 
in  tuberculosis,  and  in  infantile  marasmus  and  atrophy.  It  has  also  been 
recommended  in  cancer. 

5.  The  Thyroid  Glands. — This  gland  consists  of  two  lobes  situated  in 
the  neck  in  front  of  the  trachea.  This  is  perhaps  the  best  known  of  the 
ductless  glands  and  has  received  a  great  deal  of  attention  on  the  part 
of  special  investigators  and  clinicians.  The  congenital  absence  of  this 
gland  or  an  arrested  development  in  infancy,  is  followed  by  defective 
mental  as  well  as  physical  development.  The  body  is  small  and  more  or 
less  irregular  in  contour,  the  face  appears  swollen,  the  eyelids  puffy,  nose 
broad  and  flat,  protruding  tongue  (or  tendency  to  protrude),  abdomen 
swollen,  short  extremities,  mental  dullness  and  stupidity  amounting  to 
idiocy  in  many  cases. 

Degeneration  of  the  thyroids  in  the  adult  is  followed  by  a  complex 
group  of  symptoms,  the  most  prominent  of  which  are  thickening  of  the 
skin  due  to  hyperplasia  of  the  subcutaneous  connective  tissues  of  an 
embryonic  type  rich  in  mucinoid  material,  to  which  the  term  myxcedema 
is  applied.  As  the  result  of  these  changes  in  the  skin,  the  face  becomes 
broadened,  swollen  and  puffy.  The  features  become  irregular,  coarse 
and  expressionless.  The  mind  becomes  clouded,  memory  is  defective, 
dementia  follows  terminating  in  idiocy. 

The  general  conditions  due  to  hypothyroidism  are  designated  by  the 
terms  myxoedema,  cretinism  and  cachexia  strumipriva.  The  numerous 
cases  of  mental  and  physical  degeneracy  in  Crete  are  due  to  the  inter- 
marriage of  those  afflicted  with  hypothyroidism,  and  from  which  the 
term  cretinism  is  derived.  Both  the  cretins  as  well  as  the  form  of  degen- 
erates designated  as  mongols  (Mongolian  and  Lorain  types  of  degeneracy) 
are  due  to  inherited  or  early  hypothyroidism. 

In  excessive  secretion  of  the  thyroids  (hyperthyroidism)  there  arise 
the  symptoms  designated  under  goiter  and  exophthalmic  goiter  (Graves's 
disease,  Basedow's  disease),  represented  by  rapid  action  of  the  heart 
(tachycardia),  active  pulsation  of  the  arteries  at  the  base  of  the  neck, 
protrusion  of  the  eyeballs,  fine  tremors  of  the  hands,  and  a  more  or  less 
changed  mental  state  or  condition.  The  excessive  enlargement  of  the 
thyroid  glands  resulting  in  the  symptom  complex  laid  to  hyperthyroidism, 

19 


TOO  PHARMACEUTICAL   BACTERIOLOGY 

in  adults,  is  by  some  stated  to  be  due  to  the  drinking  water  supply,  but 
this  has  not  yet  been  satisfactorily  demonstrated  as  a  fact. 

The  thyroid  hormone  is  an  organic  iodine  compound  (colloidal  iodine) 
called  iodo-thyrin  or  thyroiodine) ,  said  to  contain  from  0.33  to  i.oo  per 
cent  iodine.  This  is  a  very  stable  compound  and  has  been  used  with 
some  success  in  hypothyroidism.  It  would  naturally  be  contraindicated 
in  hyperthyrodism. 

6.  The  Parathyroid  Glands. — These  are  supposed  to  be  accessory  thy- 
roid bodies  and  lie  near  the  latter  in  the  neck  to  the  front  and  sides  of  the 
tracheae.     The  parathyroids  as  well  as  the  thyroids  are  essential  to  life, 
as  total  extirpation  is  followed  by  death.     By  some  investigators  the 
parathyroids  are  looked  upon  as  immature  thyroid  tissue  and  there  is 
no  doubt  as  to  the  close  functional  relationship  of  the  two  glands.     Extir- 
pation of  the  parathyroids  is  followed  by  the  symptoms  designated  by  the 
term  tetany,  namely  restlessness,  excitability,  muscular  tremors,  which 
before  death,  develop  into  convulsions,  rigor  and  complete  exhaustion. 
According  to  Macallum  the  symptoms  above  cited  may  be  relieved  at 
once  by  the  intravenous  injection  of  a  soluble  calcium  salt.     In  fact  the 
prompt  effects  are  most  striking  and  has  suggested  the  use  of  calcium  salts 
in  dys-function  and  hypo-function  of  the  parathyroids. 

In  hypothyroidism  of  adults  there  is  disturbed  nerve  function,  mostly 
sympathetic,  worry,  loss  of  mental  balance,  excitability,  etc. 

7.  The  Suprarenal  or  Adrenal  Glands. — As  the  name  implies,  these 
glands,  two  in  number,  are  associated  with  the  kidneys,  one  each  lying 
just  above  the  kidney.     They  are  flattened,  more  or  less  triangular  and 
weigh  about  four  grams  each.     An  enormous  amount  of  work  has  been 
done  in  regard  to  these  glands  and  our  information  concerning  their  func- 
tional activities  is  fairly  complete.     Brown-Sequard  demonstrated  that 
removal  of  these  glands  resulted  in  death  almost  immediately  (within  a 
few  hours  to  possibly  two  or  three  days).     The  symptoms  preceding 
death  are  great  prostration,  muscular  weakness  and  diminished  vascular 
tone.     The  symptoms  resemble  those  of  Addison's  disease  which  is  now 
known  to  be  due  to  pathological  lesions  of  these  glands.     One  of  the  active 
principles  of  the  adrenals  is  the  animal  alkaloid  epinephrine,  now  a  much 
used  medicine  and  fully  described  in  all  texts  on  materia  medica.     Epine- 
phrine does  not  however  represent  the  full  physiological  action  of  the 
gland. 

In  hypo-function  of  the  adrenals  there  is  a  feeling  of  lassitude  (Spring 
fever),  liver  spots  appear  and  there  may  be  slight  general  pigmentation. 
In  decided  dys-function  of  the  adrenals  the  symptoms  deepen,  there  is 
great  loss  of  activity,  the  skin  becomes  decidedly  darkened  and  bronzed, 
which  discoloration  is  extended  to  the  mucous  membranes,  there  is  loss 


ADKN'OLOGY.      THE  ENTXXTRINOUS  GLANDS  AND  THEIR  EXTRACTS      2QI 

of  appetite,  reduced  tonicity  of  muscle  and  of  the  nerves,  disturbance  of 
the  digestive  function,  low  blood  pressure,  subnormal  temperature,  and 
feeble  respiration.  A  very  common  complication  of  Addison's  disease 
and  of  dys-function  generally,  is  general  tuberculosis  which  is  believed 
to  have  its  origin  in  the  adrenals.  This  led  to  the  supposition  that 
these  glands  secreted  an  antitoxin  or  antibody  against  tuberculosis. 

The  correlative  activities  of  the  thyroids  and  the  adrenals  are  very 
definitely  demonstrated.  The  secretions  of  the  adrenals  constrict  the 
blood  vessels  (capillaries)  while  the  thyroid  secretions  dilate  them,  adrenals 
retard  digestion  while  the  thyroids  promote  this  function,  thyroids  in- 
crease the  heart  action  while  adrenals  retard  it, 

PRESERVATION  AND  STORAGE  OF  BIOLOGIC  PRODUCTS 

Biological  products  (sera  of  all  kinds,  vaccines,  bacterins  and  glan- 
dular extracts,  etc.)  are  organic  in  nature  and  are  readily  decomposed. 
Some  kinds  of  biologies  are  more  readily  decomposed  than  others.  Some 
retain  their  physiological  and  therapeutic  properties  for  comparatively 
long  periods  of  time,  provided  they  are  properly  stored  (as  diphtheria 
antitoxin),  while  others  deteriorate  quite  rapidly  even  when  kept  under 
ideal  conditions  (as  lutein,  pollen  extracts,  and  antirabic  vaccine). 

The  chief  factors  which  hasten  the  deterioration  of  biologies  are: 

1.  Sunlight,  More  Specifically  the  Actinic  Rays.     Therefore,  biologies 
must  be  kept  in  the  dark. 

2.  Air,  the  Oxygen  of  the  Air.     The  manufacturer  protects  most  of  the 
biologies  against  this  factor  by  placing  the  suitable  amounts  in  hermet- 
ically sealed  containers.     Under  no  circumstance  are  containers  to  be 
opened  until  the  preparation  is  to  be  used,  and  then  only  by  the  physi- 
cian, under  proper  conditions. 

3.  Temperature.     Biologies    are    very    susceptible     to    temperature 
changes.    It  is  claimed  that  freezing  does  not  cause  deterioration  of  the 
products  and  apparently  this  is  well  substantiated  by  numerous  observers. 
It  would  appear,  however,  that  a  biologic  which  has  been  frozen  for  a  time 
will  deteriorate  more  rapidly  should  it  subsequently  be  exposed  to  the 
unfavorable  conditions  of  light,  air,  etc.,  as  compared  with  a  biologic 
which  had  not  been  frozen  but  otherwise  similarly  kept  and  exposed. 

All  biologies  are  quite  rapidly  decomposed  and  rendered  useless  by 
higher  temperatures  (60°  C.  and  up).  Therefore,  all  biologies  should  be 
kept  on  ice  all  of  the  time,  until  wanted  for  use. 

4.  Moisture.     Many,  but  not  by  any  means  all,  biologies  are  in  con- 
tainers which  exclude  outside  moisture.     Such  biologic  preparations  as 
are  in  the  form  of  dry  extracts  or  dry  vaccines,  or  tablets  and  pellets, 
should  be  kept  away  from  moisture.     A  safe  rule  to  follow  is  to  keep  all 


2 Q2  PHARMACEUTICAL  BACTERIOLOGY 

biologies  in  a  dry  place.  The  keeping  and  storing  of  biologies  may  be 
summarized  as  follows: 

Keep  in  a  dark  place  at  a  uniform  temperature  near  freezing. 

Bio  ogics  are  rapidly  coming  into  use  more  and  more.  Most  of  them 
are  in  the  nature  of  emergency  remedies,  desired  at  once  and  full  thera- 
peutic effects  expected  without  fail.  Only  too  frequently  does  the  physician 
fail  to  get  the  expected  effects,  simply  because  the  particular  product 
was  rendered  inert  through  improper  storing.  Only  a  small  percentage 
of  practising  pharmacists  know  what  biologies  really  are  and  how  they 
are  prepared  and  why  they  must  be  kept  thus  and  so.  In  many  of  the 
outlying  rural  districts  in  particular,  it  is  altogether  too  common  practice 
to  overstock  or  to  keep  biologies  until  they  are  entirely  worthless.  Par- 
ticularly does  this  apply  to  smallpox  vaccine. 

It  would  be  most  desirable  to  establish  conveniently  located  storage 
stations  for  biologies,  in  charge  of  experienced  and  expert  keepers.  This 
store  room  should  have  double  walls  on  all  sides  with  intervening  air 
spaces,  should  be  suitably  lighted,  well  ventilated,  and  provided  with 
artificial  refrigeration  (to  take  the  place  of  the  ice  chest).  The  slight 
differences  in  temperature  of  the  different  parts  of  the  store  room,  should 
be  utilized  to  the  best  advantage  by  the  keeper.  In  lieu  of  such  central 
stations,  the  pharmacist  must  provide  the  proper  storage.  During 
the  summer  months  (in  states  where  high  summer  temperatures — from 
70°  F.  to  110°  F. — prevail)  the  biologies  may  be  kept  in  a  special  compart- 
ment of  the  soda  fountain  refrigerator.  Certain  products  may  be  stored 
in  the  cellar  or  basement.  Some  are  kept  on  the  store  shelves,  or  behind 
the  prescription  counter,  although  this  is  objectionable  for  reasons  already 
given. 

How  long  will  biologies  keep?  Very  naturally  this  question  cannot 
be  answered  definitely,  as  so  much  depends  upon  the  varying  conditions 
already  mentioned.  In  a  general  way  no  biologic  product  should  be 
used  which  is  two  years  old,  or  more,  and  very  few  become  worthless  in 
less  than  one  month's  time.  The  following  is  a  list  of  the  more  important 
biologies,  giving  them  approximately  in  the  order  of  their  keeping 
qualities. 

1.  Diphtheria  antitoxin.     Coagulose. 

2.  Other   sera — antistreptococcic,   antimeningococcic,  antigonococcic. 

3.  Dry  glandular  extracts  and  powdered  glandular  substance. 

4.  Bacillary   tablets,   lactone   tablets,   tuberculin   tablets,   and   other 
dry  bacillary  cultures. 

5.  Ordinary  bacterins,  tuberculins. 

6.  Prophylactic  bacterins.     Sensitized  bacterins. 

7.  Smallpox  vaccine,  antirabic  vaccine,  luetin,  mallein,  etc. 


ADENOLOGY.      THE  ENDOCRINOUS  GLANDS  AND  THEIR  EXTRACTS      293 

8.  Normal  serum  against  hemorrhage.     Hemoplastin? 

9.  Liquid  pollen  extracts. 

Hydrated  biologies  (liquid  proteins,  protein  sols)  deteriorate  more 
rapidly  than  dehydrated  biologies  (dry  proteins,  protein  gels),  and  on 
first  consideration  it  would  appear  desirable  to  use  these  products  in  the 
dry  state,  but  since  they  are  to  be  administered  hypodermically  or  intra- 
venously, they  must  be  in  condition  for  immediate  absorption  and  assimi- 
lation (hence,  in  the  form  of  protein  suspensoids  or  sols).  A  protein 
which  has  been  dehydrated,  that  is,  which  has  been  changed  from  a  sol 
to  a  gel,  is  often  not  readily  reconverted  into  a  sol.  Furthermore,  the 
physician  cannot  as  a  rule,  take  the  time  to  prepare  it  properly. 

Every  pharmacist  and  also  every  physician,  should  be  familiar  with 
the  consistency,  color  and  odor  of  the  biologies,  in  their  normal  state. 
The  physician  should,  for  example,  not  use  adrenalin  which  has  become 
pinkish  in  color,  the  normal  being  colorless.  Glandular  extracts  deepen 
in  color  with  age  and  develop  an  animal  odor.  Normally  clear  prepara- 
tions which  have  become  turbid  or  flocculent,  or  which  show  a  precipitate, 
should  not  be  used.  It  is  true  that  the  manufacturers  observe  every 
precaution  to  insure  against  mishaps  and  all  products  are  tested  and  exam- 
ined before  they  are  sent  out,  yet  the  physician  as  well  as  the  pharmacist 
should  be  qualified  to  judge  of  the  quality  and  purity  of  the  preparations. 


CHAPTER  XIII 
YEASTS  AND  MOLDS 

The  prganisms  commonly  designated  as  yeasts  and  molds,  though  not 
belonging  to  the  bacteria  (Schizomycetes),  are  of  the  greatest  importance 
in  human  economy  and  play  a  most  active  part  in  life.  Some  of  them 
are  most  beneficent  while  others  are  very  injurious  to  health.  The  yeast 
organisms  (Saccharomyces)  cause  the  alcoholic  fermentations  in  saccharine 


FIG.  69. — Development  of  Mucor  mucedo.  a,  b,  c,  d,  stages  in  the  formation  of  the 
zygospore;  d,  mature  zygospore;  e,f,  endospore  formation;  g,  endospores;  h,  germinating 
spore,  this  develops  and  finally  gives  rise  to  new  zygospores;  *,  mucor  slightly  magnified. 
This  mould  is  found  on  stale  bread,  damp  leather,  gloves,  etc. 

solutions.    Many  of  the  mold   group  cause  skin   and   other  diseases. 
They  all  belong  to  the  plant  division  fungi.     The  more  important  species 
may  be  grouped  under  three  orders,  as  follows: 
I.  Phycomycetes.     (Zygomycetes.)     Zygospore  formation. 

1.  Mucor  corymbifer.  \ 

y   ,         \  Both  are  found  in  tissue  infections. 

2.  Mucor  mucedo.       J 

294 


YEASTS   AND   MOLDS 


295 


3.  Other  species  of  Mucor  are  reported  as  causing  pathologic  con- 
ditions in  man  and  in  lower  animals.     Some  are  the  cause  of  fatal  infectious 
diseases  in  such  household  pests  as  the  common  fly.    Others  attack  fruits, 
as  pears,  figs  in  particular,  leather  goods  as  gloves,  etc. 
II.  Ascomycetes.     Spores  formed  in  asci  (sacs). 
i.  Saccharomycetes — yeasts  proper. 

a.  Saccharomyces  cerevisea.  This  name  is  applied  to  many 
species  or  varieties  of  yeasts  concerned  in  fermentation 
processes,  as  in  beer,  wine  and  sake  making. 


FIG.  70. — Saccharomyces  cerevisece. 


The  form  or  variety  known  as  brewers'  top  yeast. 
(Oberhefe.) 


b.  Saccharomyces  angina.    Pathogenic. 

c.  Saccharomyces  ellipsoides.    Common  in  fermenting  fruits, 
jams,  jellies,  fruit  juices,  etc.    Other  species  are  active 
in  various  vegetable  food  fermentations. 

d.  Saccharomyces  Blanchardi.    Pathogenic. 

e.  Endomyces  albicans.     Pathogenic,  causes  thrush. 

f.  Cryptococcus  Gilchristi.     Pathogenic;  general  infections. 

g.  Cryptococcus  hominis.    Pathogenic. 
2.  Gymnoascomycetes. 

a.  Trichophyton  tonsurans.     Pathogenic,  causes  scalp  dis- 
ease (ringworm),  also  attacks  other  external  tissues. 


296 


PHARMACEUTICAL  BACTERIOLOGY 

b.  Trichophyton   Sabourandi.    Pathogenic.    Attacks    scalp 
and  beard  (ringworm). 

c.  Trichophyton  molaceum.    Pathogenic.    Like  (b).     Violet 
color. 

d.  Trichophyton    mentagrophytes.      Pathogenic.        Causes 
beard  and  body  ringworm. 

e.  Trichophyton  cruris.    Pathogenic. 

f .  Microsporum  A  udouini.    Pathogenic. 


FIG.  71. — Saccharomyces  cereviseoe.  The  form  or  variety  known  as  brewers'  bottom 
yeast.  (Unterhefe).  a,  Spore  formation;  b,  elongated  cells,  which  develop  under  certain 
conditions  of  moisture,  food  supply,  etc. 

g.  Achorion  Schosnleini.    Pathogenic.     Is  the  cause  of  that 

very  common  scalp  disease  of  children  known  as  favus. 
3.  Carpoascomycetes. 

a.  Penicillium    crustaceum.    This    is    a    blue-green    mold 
which  is  believed  to  be  pathogenic  in  chronic  catarrhal 
conditions  of  the  Eustachian  tubes  and  of  the  stomach. 

b.  Penicillium  glaucum.     This  is  the  omnipresent  blue-green 
mold  so  common  in  the  household,  infesting  all  exposed 
moist  organic  substances.     Supposed  to  be  non-patho- 


YEASTS   AND   MOLDS 


297 


genie,  although  some  credit  it  with  being  the  cause  of 
pellagra. 

c.  Aspergillus  fumigatus.     Said  to  be  the  cause  of  pellagra. 

d.  Aspergillus  concentricus.     Causes  ringworm.     Common 
in  the  Malay  peninsula,  China  and  in  the  Philippines. 
Limited  to  tropical  countries. 

d.  Aspergillus  flavus.     Pathogenic.     Found  in  chronic  dis- 
charges from  ear. 

e.  Aspergillus  repens.     Much  as  (d). 


FIG.  72. — Saccharomyces  ellipsoides.  Very  common,  in  fruit  products  as  jams,  jellies, 
etc.  Living  yeast  cells  show  budding  of  cells  and  vacuoles.  Dead  yeast  cells  usually 
occur  singly,  the  vacuoles  are  wanting  and  the  cell  walls  are  more  distinct,  generally  due 
to  the  absorption  of  coloring  substances  from  the  medium  in  which  they  occur. 

f.  Aspergillus     piclor.     Pathogenic.     Occurs     in     Central 
America,  where  it  causes  a  mange  disease. 

g.  Aspergillus    oryza.     Nonpathogenic.     Cultures    of    this 
fungus  are  used  in  the  manufacture  of  sake  (Chinese  and 
Japanese  rice  wine).     The  fungus  growing  and  feeding 
upon  ,the  steamed  rice  grains  converts  the  starch  into 
saccharine  substances  which  are  then  acted  upon  by  the 
yeast  ferment. 

III.  Hyphomycetes.     Systematic   position   of   the  pathogenic  members 
not  well  denned.    Life  history  not  yet  fully  worked  out. 

i.  Discomyces  bovis.  (Actinomyces).  The  so-called  ray  fungus 
which  causes  the  condition  in  cattle  known  as  actinomycosis,  a  disease 
which  can  be  transmitted  to  man. 


298 


PHARMACEUTICAL  BACTERIOLOGY 


2.  Discomyces  madurce.     (Mycetoma).     Causes  the  cattle  disease 
known  as  madura  foot,  which  can  be  transmitted  to  man.     Essentially  a 
tropical  disease.    Two  varieties  (black  and  white)  of  the  disease  are 
reported. 

3.  M alassezia  furfur.    This  is  the  fungus  which  causes  a  skin  dis- 
ease (Tinea  versicolor)  which  is  quite  common  in  tropical  as  well  as  in 
temperate  climates. 


PIG.  73. — Three  terminal  hypae  showing  the  characteristic  spore  formation  of  Peni- 
cillium  glaucum.  This  fungus  is  a  true  saprophyte  and  is  never  found  on  living  fruits  or 
vegetables.  Mouldy  food  substances  are  quite  universally  rejected  as  being  unfit  for 
human  consumption. 

4.  Microsporoides   minutissimus.     Causes   a  skin   disease  known  as 
Erythrasma  or  Dhobie's  itch.    Found  in  the  tropics. 

5.  Trichosporum  giganteum.     Causes    a  disease   of  the  hair.     The 
spores  of  the  fungus  are  arranged  about  the  hair  in  a  peculiar  mosaic. 

Molds  differ  from  bacteria  in  that  they  thrive  best  in  acid  media  and 
in  that  they  are  not  so  readily  killed  by  means  of  the  usual  chemical  dis- 
infectants. Heat  (dry  as  well  as  moist)  kills  the  hyphal  structure  quite 


YEASTS   AND   MOLDS  299 

readily,  but  the  spores  are  quite  resisting,  though  less  so  than  the  spores 
of  bacteria.  They  can  be  cultured  on  potato,  on  bread,  or  on  other  organic 
food  materials  (kept  moist  in  a  moist  chamber).  The  following  medium 
is  very  satisfactory. 

Peptone i  gm. 

Maltose 4  gm- 

Agar 1.5  gm. 

Water  .  100  cc. 


FIG.  74. — Actinomyces  bovis.  Showing  the  hyphal  structure  of  this  pathogenic 
fungus.  There  are  numerous  fungi  of  the  mold  group  that  cause  local  pathologic 
conditions  of  the  skin  and  mucous  membranes. 

Mix,  dissolve,  filter,  titrate  to  reaction  +2  and  sterilize  in  the  usual  way. 
Culturing  is  usually  done  in  Erlenmeyer  flasks  (250  or  500  c.c.)  with  a 
thin  layer  of  the  medium  in  the  bottom.  Before  placing  the  mold 
material  in  the  flask  (by  means  of  a  platinum  loop)  allow  it  to  macerate 
in  60  per  cent,  alcohol  for  two  hours  which  will  kill  the  bacteria  present 
without  destroying  the  life  of  the  mold.  The  acid  reaction  of  the  me- 
dium (+2)  will,  however,  usually  prevent  bacterial  growth. 

Yeast  organisms  may  be  studied  very  conveniently  in  the  hanging 
drop.  The  development  of'  mould  may  be  observed  between  two  sterile 
slides.  Since  these  organisms  are  much  larger  than  bacteria  there  is  little 


300  PHARMACEUTICAL   BACTERIOLOGY 

difficulty  in  examining  them  under  the  low  power  of  the  microscope. 
Mount  in  water  or  in  a  weak  solution  (o.io  per  cent.)  of  caustic  potash  or 
soda.  In  looking  for  yeasts  and  molds  in  liquids,  centrifugalizing  may 
be  desirable.  Staining  methods  will  rarely  be  necessary. 

While  it  is  true  that  not  all  molds  are  pathogenic,  yet  it  must  be 
remembered  that  many  are  decidedly  so,  besides  most  of  them  are  very 
objectionable  on  account  of  the  disagreeable  moldy  odor  and  taste, 
if  for  no  other  reason.  Moldy  food  substances  are  not  fit  for  consump- 
tion and  molds  should  not  occur  in  any  of  the  pharmaceuticals,  syrups, 
soda  fountain  preparations  and  fruit  juices.  Most  of  the  yeasts  are  non- 
pathogenic.  The  common  yeast  has  even  been  used  as  an  intestinal 
disinfectant  in  typhoid  fever,  yet  no  preparations  in  the  drug  store  should 
be  allowed  to  undergo  yeast  fermentation  for  the  reason  that  the  process 
changes  the  quality  and  flavor  of  the  substances  thus  attacked.  Fruit 
pulp,  fruit  juices  and  syrups  of  all  kinds  are  peculiarly  liable  to  the  attacks 
of  the  yeast  organisms  and  every  precaution  should  be  taken  to  guard 
against  such  infection.  This  is  not  a  simple  matter  because  the  yeast 
cells  and  the  yeast  spores  are  found  everywhere  and  develop  very  readily 
in  all  saccharine,  slightly  acid  substances.  Moist  heat  sterilization  or 
pasteurization  are  the  most  effectual  means  for  preventing  yeast  fer- 
mentations. 

The  yeast  cakes  used  by  the  housewife  in  making  bread  consist  simply 
of  pure  cultures  of  Saccharomyces.  The  cakes  must  be  kept  quite  dry 
and  in  the  cold  (ice  chest)  to  prevent  decomposition.  Even  under  the 
most  favorable  conditions  they  soon  become  worthless.  As  soon  as  the 
cake  is  mixed  with  the  bread  dough  with  adequate  warmth,  the  yeast  cells 
begin  to  feed  upon  the  various  available  food  substances  present  and 
multiply  rapidly  (by  budding),  resulting  in  the  formation  of  alcohol  and 
liberation  of  CO2  gas,  which  latter  in  an  attempt  to  escape,  causes  the 
so-called  rising  of  the  bread.  If  the  dough  is  not  thoroughly  mixed,  the  gas 
liberation  is  uneven  and  the  bread  will  be  unsatisfactory,  because  there 
will  be  large  cavities  in  some  parts  of  the  loaf  and  in  other  parts  the  loaf 
will  be  solid.  Bread  must  be  baked  quickly,  after  the  rising  has  reached 
the  proper  degree,  otherwise  the  loaf  will  be  flat  and  doughy.  The  house- 
wife in  the  country  simply  prepares  sour  dough  cakes  which  take  the 
place  of  the  manufactured  yeast  cakes  used  in  the  city.  In  biscuit 
making  the  desired  CC>2  gas  liberation  is  brought  about  by  the  use  of 
baking  soda  and  sour  milk  or  by  means  of  baking  powder  alone. 

The  alcoholic  fermentation  in  the  manufacture  of  beer  is  caused  by  the 
several  varieties  and  forms  of  Saccharomyces  ceremsece  (Torula  cerevisea) . 
In  beer  making,  the  barley  grain  is  first  acted  upon  by  the  starch  enzyme 
(diastase)  which  converts  the  starch  into  maltose  (malt)  and  the  maltose 


YEASTS   AND   MOLDS 


301 


is  in  turn  converted  into  alcohol  by  the  Saccharomyces.  If  the  fermenta- 
tion product  (as  grape  wine,  apple  cider,  beer,  porter,  etc.)  is  exposed  to 
the  air  for  a  time,  the  Mycoderma  aceti  enters  and  at  once  begins  to  convert 
the  alcohol  into  acetic  acid  and  we  finally  have  vinegar.  "Hard  cider" 
is  simply  apple  wine  in  which  the  acetic  acid  fermentation  has  progressed 
to  an  advanced  stage. 

In  the  manufacture  of  the  Japanese  and  Chinese  rice  wine  (sake) 
the  maltose  fermentation  of  the  starch  (in  the  rice  grain)  is  brought  about 
by  the  Aspergillus  oryzfe  as  already  stated.  The  process  of  beer  and 
sake  manufacture  may  be  compared  as  follows: 


BEER 


SAKfi. 


i.  Material  Used 


Carefully  selected  barley  is  cleaned  in 
running  water,  then  macerated  in  water 
to  induce  germination.  Rice,  wheat  and 
other  cereals  may  be  added.  Hops  are 
used. 

2.  Diastase  Fermentation. 

During  the  germinating  process  a  fer- 
ment or  enzyme  (diastase)  is  liberated 
which  converts  the  starch  into  saccharine 
compounds.  The  ferment  is  unorganized 
(non-living)  and  is  soluble  in  water.  The 
germinating  and  fermenting  grain  consti- 
tutes the  beer  wort. 


A  good  quality  of  rice  is  thoroughly 
washed  in  cold  water,  then  softened  by  a 
steaming  process.  No  hops  used. 


Malting 

The  steamed  rice  is  spread  on  mats  and 
inoculated  with  tha  spores  and  hyphse  of 
Aspergillus  oryza.  This  fungus  liberates 
an  enzyme  (disastase)  which  converts  the 
starch  into  saccharine  substances.  The 
enzyme  produced  by  the  fungus  is  soluble 
in  water.  Fermentation  takes  place  in  a 


warm  room. 


3.  Alcoholic   Fermentation 


The  beer  wort  (Bierwiirze)  is  now  ready 
to  be  acted  upon  by  the  yeast  organis  us 
(Saccharomyces  cerevisece)  which  enter  from 
the  air  or  which  may  be  added  in  pure 
culture.  The  yeast  organisms  convert  the 
saccharine  substances  into  alcohol  and 
carbonic  acid  gas  (€02). 

The  diastase  and  the  yeast  ferments  are 
both  active  during  this  process. 


The  sake  wort  (moto)  is  prepared  by  mix- 
ing the  steamed  rice  and  fungus  (A.  oryza) 
in  vats.  Yeast  cells  (Saccharomyces  of 
sake)  enter  from  the  air  and  cause  alcoholic 
fermentation,  converting  the  saccharine 
substances  into  alcohol  and  carbonic  acid 
gas  (COj). 

The  diastase  ferment  (produced  by  A. 
oryza)  and  the  alcoholic  ferment  (Saccha- 
romyces) are  active  during  the  entire 
process. 

4.  Expressing,  Cooling,  Clarifying  and  Pasteurizing 

These  processes  are  very  closely  similar  in  beer  and  sake  brewing.  The  differences, 
if  any,  are  slight  and  pertain  to  modifications  of  methods  employed  by  different  manu- 
facturers. Preservatives,  as  salicylic  acid,  may  be  added.  Both  beverages  may  be  rein- 
forced with  alcohol.  This  is  not  generally  done  with  seke  as  the  brewers  declare  thatjthe 
addition  of  foreign  alcohol  destroys  the  characteristic  flavor  or  bouquet. 


302 


PHARMACEUTICAL  BACTERIOLOGY 


5-  Kinds  or  Brands 


Many  different  brands  varying  in  color,         Different    brands    varying  in   quality, 
taste,  alcoholic  percentage,  ash  percentage,     The  alcoholic  percentage  ranges  from  14 
etc.     The  alcoholic  percentage  ranges  from     to  18.     There  is  a  sweet  variety  (Minn) 
1.5  to  6.     The  ash  percentage  is  about  8.     and  a  white  variety  (Shird).     Ash  percent- 
age about  3,  frequently  less. 


PIG.  75. — Sake"  making.  A,  B,  Rice  eel's  entirely  filled  with  starch  granules;  C, 
rice  cells  after  steaming,  the  starch  granules  are  broken  up;  D,  rice  starch  granules  a, 
dextrinized,  6,  normal. 


6.  Use  and  Properties 

A  beverage,  usually  taken  in  compara-         Usually  taken  in  small  amounts,  pro 
tively  large  doses,  producing  a  mild  form     ducing  a  speedy,  though  transient,  form  of 
of  intoxication.  intoxication.     Taken  as  a  wine.     In  Japan 

sake*  is  usually  heated  before  drinking. 


YEASTS   AND   MOLDS 


303 


There  are  numerous  varieties  of  Saccharomyces  concerned  in  beer 
brewing.  There  are  several  kinds  of  upper  or  top  yeasts  (Kahmhefe 
Oberhefe}  and  several  kinds  of  bottom  or  lower  yeasts  ( Unterh efe) ,  each 
kind  possessing  supposedly  special  properties.  Just  what  part  the  more 
or  less  incidentally  associated  organisms  (as  bacteria,  molds,  and  foreign 


FIG.  76. — Sake  making.  Steamed  rice  cells  (c)  attacked  by  the  hyphae  (a)  of 
Aspergillus  oryzce  which  feed  upon  the  dexlrinized  rice  starch,  converting  it  into  sac- 
charine substances.  Yeast  cells  and  bacilli  are  usually  associated  with  the  hyphal  fun- 
gus, feeding  upon  the  saccharine  substances  formed. 

yeasts)  may  play  in  the  fermentation  processes  is  not  clearly  understood. 
It  is  known  that  some  of  these  extraneous  organisms  may  develop  to  such 
an  extent  as  to  modify  the  quality  of  the  product  completely.  Such  fer- 
mentation diseases  are  a  source  of  much  annoyance  to  manufacturers, 
often  resulting  in  great  financial  loss,  but  this  has  also  been  the  great 
stimulus  in  compelling  the  use  of  pure  cultures  and  in  perfecting  those 
methods  which  are  known  to  improve  the  keeping  qualities  of  the  articles, 


304 


PHARMACEUTICAL  BACTERIOLOGY 


whether  foods  or  drink.  Sake  in  particular,  does  not  keep  well,  even  with 
the  greatest  care  in  manufacture  and  with  the  use  of  preservatives.  Cer- 
tain brands  of  beer,  wine,  sake,  smoking  tobacco,  cigars,  tea,  etc.,  are 
known  to  lose  their  characteristic  flavors  within  short  periods,  due  to  the 


FIG.  77. — Sak6  making.     Aspergillus  oryzce,  showing  vegetative  hyphae  (a),  and  spore- 
forming  hyphse  (b,  c,  d). 


invasion  of  some  "disease"  producing  organism.  In  many  instances 
manufacturers  have  been  blamed  for  inferiority  in  the  quality  of  fer- 
mented products  when  in  reality  said  articles  left  the  establishment  in 
perfect  condition  as  far  as  quality  is  concerned,  but  were  subsequently 
(in  shipment,  in  storage,  etc.)  attacked  by  some  objectionable  organism, 
resulting  in  a  complete  change  of  flavor  or  bouquet. 


YEASTS   AND   MOLDS 


305 


The  Japanese  soya  sauce  (fermented  soya  beans,  Glycine  hispidus)  and 
miso,  a  soup  stock  of  wheat  and  soya  beans,  is  prepared  through  the  action 
of  Aspergillus  oryzce  and  A.  wentii.  The  Javanese  arrak  is  made  fronrriee 
which  is  first  acted  upon  by  a  fungus  (Ragi)  in  many  respects  similar  to  A. 
oryza,  and  subsequently,  the  ateoholic  fermentation  is  carried  on  by  the 
Saccharomyces,  thus  the  method  of  arrak  manufacture  is  closely  similar  to 


PIG.  78. — Sake  making.  A,  Dead  or  dying  yeast  cells;  B,  active  yeast  cells 
which  convert  the  saccharine  substances  formed  by  the  aspergillus  into  alcohol.  C,  D, 
yeast  cells  and  hyphae  of  aspergillus  from  the  fermenting  vats. 

that  of  sake.  More  generally,  however,  arrak  is  made  from  fermented  mo- 
lasses. There  are  many  other  species  of  mold,  including  the  very  com- 
mon PenicUlium  glaucum,  which  have  the  power  of  converting  starch  into 
saccharine  compounds  in  the  presence  of  moisture,  but  thus  far  these  are 
not  used  industrially.  An  alcoholic  drink  of  the  East  Indies  is  prepared 
from  a  starchy  root  as  follows:  A  number  of  people,  usually  girls,  sit 
about  a  large  vessel  masticating  the  roots  which  are  then  expectorated  into 
the  vessel.  The  ferment  ptyalin  of  the  saliva  converts  the  starch  into 
saccharine  substances  which  is  then  acted  upon  by  the  Saccharomyces, 
20 


306  PHARMACEUTICAL  BACTERIOLOGY 

resulting  in  an  alcoholic  drink  which  is  said  to  have  a  very  peculiar  flavor. 
Pressed  yeast  cakes  for  bread  making  are  prepared  as  follows: 

The  filtered  saccharine  yeast  mash  in  vats,  is  inoculated  with  pure 
cultures  of  Saccharomyces  cerevisea.  Active  fermentation  takes  place  in  the 
presence  of  pure  air  which  is  supplied  through  pipes  leading  into  the  vat. 


FIG.  79. — Showing  the  characteristic  stellate  cells  of  the  pith  of  some  reed  used  as 
filtering  material  in  clarifying  sak6.  Bundles  of  the  pith  are  placed  in  the  bottom  of 
a  perforated  cask,  forming  a  layer  a  foot  or  more  in  depth;  through  this  the  sak6  perco- 
lates. The  impurities  are  caught  in  the  intercellular  spaces  of  the  pith. 

The  white  scum  or  foam  which  forms  is  poured  on  fine  sieves,  washed 
with  sterile  water,  and*then  centrifugalized  to  remove  most  of  the  water. 
This  partially  dry  material  is  then  pressed  into  cakes,  thoroughly  dried  at 
a  low  temperature,  and  wrapped  in  lead  foil  to  exclude  air.  Starch  is  some- 
times added  as  a  dryer,  but  this  is  no  longer  necessary  because  of  the 
improved  methods  of  manufacture.  Good  yeast  should  be  of  a  yellowish 
color,  easily  powdered  and  should  have  a  pleasant  "yeasty"  odor. 

The  so-called  Chinese  yeast,  concerned  in  various  fermentation  proc- 


YEASTS   AND   MOLDS  307 

esses  is  a  mixture  of  Mucor  species,  yeasts  and  bacteria.  The  following 
species  of  mucor  are  prevalent — M.  racemosus,  M.  alternans,  M.  spinosus,  M. 
circinelloides  and  M.  Boidinii.  These  have  the  power  of  converting  starch 
into  saccharine  compounds,  which  are  then  acted  upon  by  the  Saccharo- 
myces.  Various  alcoholic  ferments  have  been  employed  in  China  and 
Japan  since  time  immemorial. 

Nuclein  is  prepared  from  yeast  and  other  vegetable  cells  and  is  very 
much  used  in  the  treatment  of  certain  diseases  due  to  pathogenic  bacteria. 
It  is  said  to  have  strong  bacteriolytic  properties  and  to  increase 
phagocytosis. 

TOXIC  YEASTS  AND  MOLDS 

Within  recent  years  numerous  outbreaks  of  cattle  poisoning  have 
occurred  which  have  been  traced  to  fodder  used.  Pammel  of  the  univer- 
sity of  Iowa  has  gathered  numerous  reports  of  occurrences  of  this  kind  and 
he  is  inclined  to  lay  the  blame  for  many  cases  of  poisoning  of  this  kind  to 
fungi.  Several  species  of  Fusarium  are  mentioned  as  being  responsible  for 
equine  diseases.  Fusarium  equinum  Norgaard,  is  said  to  cause  epidemic 
itch  among  horses.  Another  species  is  said  to  cause  fatal  meningitis  in 
cattle.  Pammel  reports  that  sorghum  cane  fodder  is  frequently  causative 
of  fatal  poisoning  among  cattle.  Many  cases  of  epidemic  poisoning  among 
cattle  have  been  laid  to  silage,  especially  numerous  are  the  case  reports 
from  the  southern  states.  Some  have  suggested  that  the  trouble  is  due  to 
botulism  (fatal  poisoning  due  to  a  toxin  formed  by  the  Bacillus  botulinus, 
the  so-called  sausage  bacillus) .  The  facts  are  that  most  of  the  cases  of 
poisoning  among  cattle  are  inadequately  investigated,  and  hasty  con- 
clusions are  often  based  upon  insufficient  data. 

A  corn  silage  fungus,  the  Monascus  purpureus,  is  supposed  to  be  re- 
sponsible for  poisoning.  Moldy  fodder  is  universally  recognized  as  being 
poisonous,  but  it  has  not  yet  been  definitely  determined  which  of  the 
several  symbionts  which  usually  occur  on  such  fodder,  are  primarily 
responsible  for  the  ill  effects.  Numerous  parasitic  Saccharomyces  have 
been  found,  in  Daphnids,  in  horses,  in  guinea  pigs,  in  pigeons,  and  in 
humans.  Higher  fungi  are  responsible  for  skin  diseases,  etc.  It  is  how- 
ever, only  recently  that  the  toxigenic  parasitic  yeasts  have  received  any 
considerable  attention. 

It  is  but  natural  to  suppose  that  a  fungus  which  has  adapted  itself 
to  parasitism  upon  animals,  must  have  undergone  extensive  physiological 
as  well  as  morphological  adaptation  changes,  and  it  is  a  fact  that  most  of 
the  fungi  of  this  kind  are  morphologically  unrecognizable  or  unidentifi- 
able and  the  tendency  is  to  place  them  in  a  separate  group,  the  so-called 
fungi  imperfecti.  As  a  rule  they  show  remarkable  life  habits  and  peculiar 


308  PHARMACEUTICAL  BACTERIOLOGY 

spore  formations.  Since  the  yeasts  are  sugar  feeders  and  vegetarian  we 
may  expect  to  find  them  in  plants  and  in  fodder  containing  more  or  less 
sugar,  and  also  for  these  reasons  we  may  expect  to  find  them  parasitically 
associated  with  herbivorous  animals,  less  commonly  with  omnivorous 
animals  and  least  of  all  with  carnivorous  animals.  One  of  the  many 
reasons  why  our  knowledge  of  food  poisoning  (in  cattle  as  well  as  in  humans) 
is  so  incomplete  is  because  most  of  the  methods  of  investigation  of  cases 
are  chemical.  Rarely  is  the  microscope  brought  into  play  and  even  when 
this  is  done,  the  work  is  left  to  amateurs  who  usually  report  negatively, 
not  because  there  is  nothing  to  report,  but  rather  because  they  fail  to 
observe  or  fail  to  recognize  the  foreign  organisms  which  may  be  present. 

The  following  descriptions  of  parasitic  Saccharomyces  will  serve  to 
illustrate  the  points  above  set  forth.  In  order  that  the  student  may 
realize  the  remarkable  life  habits  and  morphological  characteristics  of  the 
two  parasitic  yeasts,  he  should  familiarize  himself  with  yeasts  in  general. 
Such  information  may  be  gleaned  from  any  of  the  more  complete  texts  on 
general  botany  and  the  special  treatises  on  yeasts  and  on  fermentation. 

The  first  case  pertains  to  the  poisoning  of  sheep  in  a  San  Francisco 
stock  yard.  The  toxicological  examination  (chemical)  was  negative. 
The  following  is  the  report  of  the  microanalyst  who  was  called  into  the 
case. 

"  I  hereby  submit  a  report  on  the  poisoning  of  sheep  which  occurred  at 
the  San  Francisco  stockyards,  Feb.  12,  1918,  and  for  several  days  subse- 
quently. I  beg  to  state  that  certain  phases  of  the  observations  made  by 
myself  are  not  finally  conclusive.  However,  the  following  statements, 
recommendations  and  conclusions  are  warranted,  based  upon  the  tests 
and  observations  made  to  date. 

The  sheep  (some  257  in  number)  were  evidently  killed  by  a  toxin 
(poison)  formed  in  the  intestinal  tract  of  the  animals,  due  to  the  presence 
of  a  parasite  which  belongs  to  the  yeast  group  (Saccharomycetes).  The 
spores  of  this  organism,  which  are  apparently  derived  from  the  barley  as 
well  as  from  the  barley  screenings,  enter  the  stomachs  of  the  animals,  with 
the  food,  where  they  develop  into  mature  vegetable  cells,  whereupon  these 
multiply  quite  rapidly  (by  a  process  known  as  'budding').  The  toxin  or 
poison  (evidently  an  exotoxin)  is  formed  by  the  growing  and  budding 
vegetative  cells  in  the  stomach  of  the  animals.  The  parasite  next  passes 
into  the  small  intestine  along  with  the  food,  where  further  development  is 
completely  checked  (due  to  the  alkaline  reaction  and  enzymes  present). 
Spore  formation  takes  place  in  the  large  intestine  and  the  spores  escape 
with  the  excreta. 

The  spores  are  very  small,  resembling  bacteria,  and  each  spore  bearing 
cell  forms  from  fifty  to  one  hundred  and  more.  Air  currents  spread  the 


YEASTS   AND   MOLDS 


309 


dried  excreta  containing  the  spores  about,  and  those  which  lodge  upon  the 
barley  straw  and  barley  grains  (and  no  doubt  also  upon  other  cereals  and 
other  forage  plants)  on  entering  the  intestinal  tract  of  susceptible  animals 
which  may  happen  to  feed  upon  such  contaminated  material,  will  multiply 
in  the  manner  already  stated.  The  probabilities  are  that  wild  rabbits, 
rats  and  mice,  as  well  as  sheep,  cattle,  domestic  rabbits  and  guinea  pigs, 
are  the  carriers  and  disseminators  of  the  infection. 

It  is  evident  that  not  all  animals,  even  of  the  same  kind,  are  equally 
susceptible  to  the  infection  and  the  poison  formed,  as  not  all  animals  died 
which  were  fed  approximately  equal  amounts  of  the  spore  contaminated 


PIG.  80. — A  toxigenic  saccharomycete  responsible  for  the  fatal  poisoning  of  sheep. 
It  is  an  obligative  parasite  and  will  not  develop  outside  of  the  intestinal  tract  of  sus- 
ceptible animals  (sheep,  goats,  rabbits,  guinea  pigs  and  probably  other  herbivora) .  a, 
the  vegetative  cells  which  occur  in  the  stomach,  where  they  grow  and  multiply  by 
budding,  b,  the  spore  bearing  cells,  showing  numerous  transverse  markings,  some 
heavy  and  the  rest  light.  The  sporangium  breaks  across  along  the  heavier  lines  and 
the  spores  are  thus  allowed  to  escape  as  shown  at  c.  d,  a  group  of  the  small  spores, 
measuring  about  four  microns  in  length. 


material.  It  is  also  evident  that  a  definite  number  of  the  spores  must  be 
taken  in  with  the  food  in  order  to  produce  fatal  intoxication.  It  is  further 
evident  that  the  organism  in  question  does  not  grow  and  multiply  outside  of 
the  stomachs  of  living  susceptible  animals.  As  soon  as  the  animal  dies  the 
vegetative  cells  also  die,  only  the  matured  spores  surviving,  as  already 
explained.  The  barley  and  barley  screenings  themselves  contain  no  toxin 
as  was  proven  experimentally. 

A  careful  microscopical  examination  of  the  ground  alfalfa,  barley,  bar- 
ley screenings  and  "  black  strap  "  molasses  samples  submitted  to  me  proved 
the  absence  of  poisonous  weeds.  The  barley,  and  especially  the  barley 
screenings  contained  a  considerable  amount  of  barley  smut  (a  fungus 
belonging  to  the  Ustilago  genus)  but  not  enough  to  produce  fatal  poisoning 
or  even  toxic  symptoms,  and  none  of  the  symptoms  of  the  experimental 


310  PHARMACEUTICAL  BACTERIOLOGY 

animals  which  died  resembled  those  of  either  acute  or  chronic  smut 
poisoning  (ergotism) . 

While  the  " black  strap"  used  in  sheep  feeding  does  not  carry  the  infec- 
tion herein  referred  to,  it  no  doubt  serves  as  a  food  for  the  parasitic  organ- 
ism, and  it  is  advised  not  to  use  it  with  barley  screenings  or  with  any  other 
forage  material  which  may  contain  the  spores  of  the  parasite  in  question. 
The  black  strap  in  all  probability  hastens  the  growth  and  multiplication 
of  the  vegetative  cells  of  the  toxigenic  parasite. 

I  would  advise  that  the  lots  of  barley  and  of  barley  screenings  of  which 
samples  were  submitted  for  experimentation,  be  not  fed  to  horses,  cattle, 
sheep  or  rabbits.  It  is  highly  probable  that  it  might  be  fed  to  hogs  with 
impunity,  as  these  animals  are  comparatively  immune  to  most  toxic  and 
toxigenic  foods.  I  would  also  advise  the  following: 

1.  That  barley  screenings  be  not  fed  to  sheep,  young  cattle,  goats  or 
rabbits.     It  would,  in  fact,  be.  inadvisable  to  feed  this  material  to  any 
animals  excepting,  perhaps,  hogs. 

2.  A  new  lot  of  barley  or  barley  screenings  should  be  tested  as  follows 
before  feeding  it: 

(a)  Feed  the  barley  or  barley  screenings   to  several  young  rabbits 
(these  animals  are  quite  susceptible  to  the  poison).     If  one  or  more  die 
within  10  to  48  hours  the  article  in  question  should  be  rejected. 

(b)  If  none  of  the  experimental  rabbits  die  within  48  hours,  feed  a 
reasonable  allowance  to  several  heads  of  sheep,  and  if  no  deaths  or  illness 
results  within  48  hours,  the  article  in  question  may  then  be  fed  to  the  entire 
herd,  allowing  rather  scant  portion  at  first. 

Among  the  causes  which  have  been  suggested  as  being  responsible 
for  the  death  of  the  sheep  are: 

1.  A  cute  Gastritis. — The  autopsies  showed  no  evidence  of  such  disease. 

2 .  Chemical  Poisons. — The  findings  of  the  city  toxicologist  were  negative. 

3.  Added  Poisonous  Weeds. — The  microscopical  findings  were  wholly 
negative. 

4.  Botulism. — Symptoms  not  those  of  botulism. 

5.  Ergotism. — Symptoms  not*  those  of  ergot  or  smut  poisoning. 

6.  Bloating. — No  evidence  of  bloating. 

7.  Use  of  Fermented  Barley. — Barley  and  screenings  in  question  showed 
no  signs  of  ever  having  undergone  fermentation. 

8.  Overfeeding. — Denied    by  Taafe  and  Co.  and  autopsies  showed  no 
evidence  of  overfeeding. 

9.  Alfalfa. — Control  tests  proved  that  the  alfalfa  used  (dried,  shredded 
or  ground  alfalfa)  was  not  poisonous. 

10.  Green  Alfalfa. — The  poisoned  animals  were  not  fed  green  alfalfa. 
The  details  of  the  experiments  and  observations  upon  which  this 


YEASTS    AND    MOLDS 


311 


report  is  based,  including  a  full  description  of  the  toxigenic  parasite,  will 
be  given  in  a  later  report." 

A  saccharomycetous  ascomycete  (N ematospora  Lycopersici,  n.-sp^ 
was  found  on  some  ripe  tomatoes  obtained  from  a  Berkeley  (California) 
restaurant.  The  tomatoes  were  from  a  lot  in  cold  storage  which,  so  it 
was  claimed,  were  imported  from  the  South  Sea  Islands.  The  specimens 


FIG.  8 1 . — Various  forms  of  vegetative  cells  of  N  ematospora  Lycopersici.  Extremes  in 
cell  formation  are  not  shown.  Very  frequently  some  of  the  hyphal  filaments  resemble 
the  hyphae  of  true  molds,  but  the  individual  cells  do  not  branch.  B,  arthrospores;  C, 
the  beginning  of  spore  sac  formation. 

under  consideration  appeared  normal  with  the  exception  of  an  area  about 
%  inch  in  diameter.  This  area  was  slightly  depressed,  of  a  cancerous 
raw  reddish  color.  The  epidermal  tissue  appeared  markedly  indurated 
and  somewhat  shrunken,  but  the  hypodermal  tissue  as  well  as  the  paren- 
chymatous  tissue  underneath  appeared  to  be  nearly  normal.  The  micro- 
scopical examination  showed  the  presence  of  a  fungus  in  the  seed  chamber 
and  in  the  mucilaginous  tissue  surrounding  the  seeds,  as  well  as  in  the 
parenchymatous  tissue  beneath  the  epidermis. 

This  fungus  proved  of  special  interest  because  every  slide  mount 
examined,  showed  the  complete '  life  cycle  of  the  organism,  including  the 
formation  and  development  of  the  polymorphic  vegetative  cells  and  the 

1  Albert  Schneider.  A  Parasitic  Saccharomycete  of  the  Tomato.  Phytopathology. 
6:395-399- 


312 


PHARMACEUTICAL  BACTERIOLOGY 


various  stages  of  gametic  fusion  and  of  ascospore  formation  and  the  forma- 
tion of  Arthrospores.  The  vegetative  cells  increased  numerically  by 
budding  as  typified  by  the  saccharomycetes  generally.  The  normal 
vegetative  cells  may  be  described  as  elliptical  to  distinctively  egg  shaped, 
without  vacuoles  and  without  recognizable  nuclei.  The  plasmic^ubstance 
lines  the  inner  wall  of  the  cell  and  is  more  abundant  at j  one  end  of  the 
cells,  usually  the  distal  end  in  case  of  cell  aggregates.  £  The  (vegetative 


FIG.  82. — Various  stages  in  gametic  fusion  and  spore  formation  of  nematospora.  A, 
two  somatic  or  vegetative  cells  unite  end  to  end  with  solution  of  the  contact  cell  walls; 
B,  the  plasmic  contents  of  the  two  cells  fuse;  C,  the  fused  plasm  becomes  grouped  into 
four  masses;  D,  plasmic  differentiation  has  proceeded  to  the  formation  of  the  eight  spore 
forming  plasmic  masses  which  soon  draw  away  from  the  wall  of  the  spore  sac  and  occupy 
a  middle  position  in  the  spore  sac  (ascus);  E,  fully  formed  spores;  F,  the  ascus  wall 
soon  dissolves  setting  free  the  eight  spores  in  two  groups  of  four  spores  each  which 
remain  attached  to  each  other  by  means  of  the  whip-like  appendages;  G,  gradually 
the  spores  become  separated  and  are  distributed  through  the  medium. 

cells  may  however  undergo  remarkable  changes  in  form.  They  may 
become  greatly  elongated,  narrowed  or  widened.  Occasionally  a  cell 
may  become  bent  or  elbowed,  narrowed  at  one  end  and  enlarged  at  the 
other  (gourd  form).  Daughter  cells  are  always  developed  apically,  never 
laterally  as  in  many  of  the  true  Saccharomycetes.  The  exceptions  are 
the  cell  formations  at  the  junctures  of  two  cells.  Daughter  cell  forma 
tion  is  bipolar,  that  is  starting  with  a  single  vegetative  cell,  new  cells  may 
form  from  the  two  apices  and  this  is  in  fact  the  rule. 

The  plasmic  contents  of  all  cells  inclusive  of  ascospores  and  of  arthro- 


YEASTS   AND   MOLDS 


313 


spores,  appear  to  be  homogeneous  with  the  exception  of  a  comparatively 
small  number  of  large  spherical  0.5  micron  granules.  The  plasmic  gran- 
ules are  especially  prominent  and  numerous  in  the  arthrospores  and- 
in  the  arthrospore  sphaerocytes.  They  are  highly  refractive  and  stain 
readily.  They  are  actively  motile,  especially  in  the  sphaerocytes  where 
they  also  show  remarkable  Brownian  vibration. 


FIG.  83. — Arthrospore  formation  and  ascospore  development  of  Nematospora. 
Ordinary  vegetative  (somatic)  cells  are  gradually  transformed  into  spores;  A,  arthro- 
spores derived  from  vegetative  cells;  B,  an  ascospore  entering  upon  a  new  vegetative 
cycle;  C,  detailed  structure  of  a  mature  ascospore  more  highly  magnified;  a,  vacuolesin 
the  chromatin-bearing  cell  of  the  spore;  6,  chromatin  substance;  c,  transverse  septum; 
d,  plasmic  masses  in  the  achromatin  cell  of  the  spore;  e,  achromatin ; /,  the  portion  of  the 
Ugule  which  stains  very  heavily;  g,  the  greatly  elongated  ligule  by  means  of  which  the 
spore  attaches  itself  to  various  substances  with  which  it  is  brought  in  contact.  The 
ascospore  is  distinctly  two-celled.  The  spore  wall  as  well  as  the  septum  are  thin. 
There  is  a  distinct  widening  of  the  spore  at  and  near  the  transverse  septum. 

Ascospore  formation  is  generally  the  result  of  the  gametic  union 
(isogamous)  of  two  elliptical  vegetative  cells.  The  apical  cell  membranes 
which  are  in  contact  dissolve.  The  plasmic  contents  of  the  two  cells  fuse. 
The  complete  changes  are  shown  in  Fig.  81.  Eight  spores  are  formed  in 
each  spore  sac.  At  an  early  period  in  the  development  of  the  spore 
sac  (ascus)  the  associated  vegetative  cells  become  separated  and  the  spore 
sac  exists  as  an  independent  cell  structure.  There  are  indications  that  a 
spore  sac  may  be  derived  from  a  single  vegetative  cell,  especially  when 
spore  formation  becomes  very  active. 

As  a  rule  active  ascospore  formation  is  accompanied  by  active  arthro- 


314 


PHARMACEUTICAL  BACTERIOLOGY 


spore  formation.  Arthrospores  are  simply  enlarged  vegetative  cells 
which  as  a  rule  assume  the  spherical  form  with  thickening  of  the  cell-wall. 
As  a  rule  the  arthrospores  also  become  separated  from  the  vegetative 
cells.  Occasionally  two  or  three  vegetative  cells  in  one  group  may  be- 
come transformed  into  arthrospores  and  remain  united  for  a  time.  Occa- 
sionally arthrospores  take  on  the  gourd  form  as  shown  in  Fig.  83. 

The  ascospores  are  two-celled,  rather  slender  and  tapering  pointed 
The  two  cells  differ  materially.  The  end  which  is  directed  toward  the 
middle  of  the  spore  sac  stains  very  heavily  and  has  a  long  slender  gelatinous 
ligule  or  filament  which  is  motionless.  This  filament  serves  to  attach  the 


FIG.  84. — Phases  jin  the  development  of  the  arthrospores  of  Nematospora  (A); 
B,  dying  spores  as  indicated  by  plasmolysis;  C,  sphaerocyte  formation  in  the  spore. 
The  mature  arthrospores  always  take  a  bipolar  position  in  the  liquid  medium,  so  that 
the  plasmic  granules  always  appear  in  profile  relative  to  the  observer. 


spore  to  its  fellows  and  to  other  objects  with  which  the  filament  may 
come  in  contact.  Gradually,  when  the  spore  begins  a  new  cycle  of  cell 
formation  by  budding,  the  ligule  disappears.  There  are  indications  that 
the  chromatin  cell  of  the  spore  serves  as  a  source  of  food  supply  for 
the  achromatin  cell  which  is  chiefly  concerned  in  starting  a  new  cycle  of 
cell  formation.  The  chromatin  cell  of  the  spore  gradually  shrinks  and 
the  unused  portions  of  the  cell-contents  disintegrate  and  dissolve  in  the 
surrounding  medium. 

The  fungus  is  typically  parasitic  in  habit  and  dies  with  the  invasion  of 
mold  and  of  rotting  bacteria,  preceded  by  very  active  asco-  and  arthrospore 
formation.  Spore  bearing  material  injected  into  a  guinea  pig  was  without 
notable  results. 


YEASTS   AND   MOLDS  315 

As  decay  of  the  tomato  advances  due  to  the  invasion  of  rotting  bacteria, 
all  vegetative  cell  multiplication  of  the  parasite  ceases.  Gradually  asco- 
spore  formation  ceases  also  and  the  existing  vegetative  cells  become  trans- 
formed into  unusually  large,  absolutely  spherical  arthrospores.  These 
arthrospores  resemble  sphaerocytes  very  closely.  Each  cell  contains  a 
homogeneous  nucleus  of  very  indistinct  irregular  outline  with  a  more  dis- 
tinct but  also  homogeneous  spherical  nucleolus.  The  plasmic  granules 
are  unusually  large,  spherical  in  form,  highly  refractive  and  stain  readily. 
They  are  slightly  motile  and  as  a  rule  occur  in  pairs,  resembling  diplococci. 
These  plasmic  granules  occur  on  the  exterior  of  the  homogeneous  plasmic 
supporting  substance  in  this*  regard  differing  from  the  plasmic  granules  of 
chlorophyll  bearing  plants  in  which  they  occur  within  the  plasmic  substance. 

The  arthrospores  occupy  a  definite  position  in  the  medium  in  which 
they  live  (the  liquid  of  the  tomato).  The  nucleus  always  occupies  such 
a  position  as  to  bring  the  edge  of  the  nucleus  into  view.  Very  rarely  the 
spore  is  sufficiently  tilted  to  present  the  view  indicated  in  (A),  Fig. 
84.  In  not  a  single  instance  was  the  spore  to  be  seen  in  exactly  vertical  view. 
No  explanation  is  offered  as  to  why  the  spore  should  assume  this  very 
definite  position. 

Plasmic  granules  are  sparingly  present  in  the  vegetal  ive  cells,  from  one 
or  two  to  five  whereas  in  the  arthrospores  there  may  be  as  many  as  one 
hundred.  As  the  arthrospores  die  the  plasmic  granules  apparently 
increase  in  number  somewhat  as  some  show  a  remarkable  increase  in  size 
and  they  become  absolutely  motionless.  All  Brownian  vibration  ceases 
also.  The  plasmic  supporting  substance  shrinks,  thus  bringing  the  granules 
closer  together  until  there  is  finally  a  closely  crowded  mass  of  plasmic  gran- 
ules as  shown  in  (B),  Fig.  84. 


CHAPTER  XIV 
PROTOZOA  IN  DISEASE 

Certain  low  forms  of  animal  life  are  causative  of  such  diseases  as 
malaria  and  sleeping  sickness.  These  organisms  resemble  each  other  in 
that  they  are  minute,  of  simple  structure  (single  celled)  and  in  that  they 
show  active  motion  due  to  the  presence  of  pseudopodia,  of  flagellae  or 
cilia,  or  due  to  cell  undulations.  They  are  found  in  stagnant  water  con- 
taining decaying  vegetable  and  animal  matter  and  in  decaying  organic 
matter.  Most  of  them  are  non-pathogenic  and  all  are  quite  readily  killed 
by  means  of  heat  and  the  common  chemical  disinfectants.  They  do  not 
occur  in  pure,  fresh  well,  spring,  or  hydrant  water. 

The  following  are  the  more  important  species  of  protozoa  and  the  prin- 
cipal activities  in  which  they  are  concerned : 

I.  RHIZOPODA. — These  move  by  throwing  out  slender  protoplasmic  pro- 
jections.    Silicious  coverings  may  be  present. 

The  amebas  form  the  type  group  of  the  Rhizopoda.  These  organisms 
are  very  widely  distributed  in  aqueous  substances  rich  in  organic  matter. 
Two  subdivisions  of  the  group  are  recognized,  the  amebas  proper  which 
feed  upon  dead  organic  matter  and  also  on  minute  organisms,  such  as 
bacteria,  and  yeasts;  and  the  entamebas  which  are  parasitic  upon  a  variety 
of  organisms,  plant  as  well  as  animal.  Even  the  parasitic  forms  feed  upon 
some  of  the  microorganisms  found  in  association  with  the  host.  The  non- 
parasitic  amebas  usually  contain  one  nucleus  whereas  the  parasitic  forms 
are  generally  multinuclear.  The  non-parasitic  forms  are  of  no  special 
significance  from  the  standpoint  of  health  and  preventive  medicine.  They 
are  scavengers  in  so  far  as  they  feed  upon  the  organisms  of  decay  and  the 
products  of  decomposition,  provided  the  water  supply  is  adequate,  for 
they  are  all  aquatic  in  habit.  If  amebas  are  abundant  in  water  supply,  it 
is  evidence  of  high  organic  contamination. 

The  parasitic  forms  no  doubt  began  existence  as  saprophytes,  gradu- 
ally changing  to  a  parasitic  mode  of  living  as  the  opportunities  for  taking  up 
the  food  materials  elaborated  by  the  host  organism  developed.  The 
entamebas  of  the  intestinal  tract  and  of 'the  mouth  also  feed  upon  the 
bacteria  and  other  microorganisms  found  in  these  localities,  but  they  also 
set  free  toxic  agents  which  give  rise  to  the  phenomena  designated  as  amebic 
dysentery,  amebic  pyorrhea,  etc. 

316 


PROTOZOA   IN  DISEASE  317 

The  amebas  are  also  classed  with  the  Sporozoa,  but  it  has  not  been 
proven  that  all  of  the  organisms  which  are  classed  as  amebas  form  swarm 
spores.  The  primary  cause  of  malaria  certainly  belongs  to  the  sporozoa. 
Cytologists  are  gradually  recognizing  the  fact  that  many  of  the  so-called 
single-celled  organisms,  such  as  ameba,  paramecium,  etc.,  are  not  as 
simple  in  structure  and  in  physiological  activity  as  was  generally  supposed. 
Many  of  these  organisms  contain  highly  complex  cell  constituents  which 
enable  them  to  compete  quite  successfully  in  the  struggle  for  existence, 
with  the  highly  complex  multicellular  organisms. 

1.  Entamosba  coli. — Inhabits  the  large  intestine.     Probably  harmless. 
Amoeba  belong  here  and  not  in  the  sporozoa.     May  be  confused  with 
phagocytes. 

2.  Entamceba  histolytica. — Causes  entero-colitis  and  dysenteric  ulcera- 
tions.     It  is  also  found  in  abscesses  of  the  liver.     Occurs  in  tropical 
countries,  less  common  in  temperate  zones. 

3.  Entamceba  buccalis. — Found  in  dental  caries.     Probably  not  patho- 
genic. 

4.  Entamosba  undulans. — Occurs  in  the  intestinal  tract. 

5.  Leydenia  gemmipara. — Identity   doubtful.     Supposed  to  have  a 
causal  relationship  to  carcinomatosis  (cancer). 

II.  FLAGELLATA. — Motion  due  to  flagellae.     Some  possess  an  undulatory 
motion.     Have  been  classed  as  bacteria  (Spirillae). 

1 .  Spirochcsta  recurrentis  (Spirillum  obermeieri) . ' — This  is  the  organism 
which  causes  relapsing  fever.     The  disease  is  so  designated  because 
after  apparent  complete  recovery,  one  or  more  relapses  invariably 
follow.    It  is  not  a  very  fatal  disease  (4  per  cent,  of  deaths)  and  is,  so  far, 
rare  in  the  United  States.     It  is  and  has  been  very  prevalent  in  parts 
of  Europe.     The  disease  can  be  transmitted,  by  inoculation,  to  man, 
monkeys,  mice  and  rats.     An  immunity  treatment  has  been  attemp- 
ted with  some  success.     Most  authorities  class  the  organism  as  a 
fungus  (Spirillum). 

2.  Spirochcsta  Duttoni. — This  organism  is  the  primary  cause  of  the 
South  African  tick  fever  (Tete  fever),  so-called  because  the  carrier  is  a 
species  of  cattle  tick  (Ornithodoras  moubata). 

3.  Spirochata  Novyi. — Said  to  be  the  cause  of  American  relapsing  fever. 

lThe  systematic  position  of  the  spirochaetes  is  still  in  dispute.  They  probably 
belong  to  the  animal  kingdom.  Note  the  tentative  position  given  them  by  the  com- 
mittee on  the  classification  of  bacteria  appointed  by  the  Society  of  American  Bacteri- 
ologists. The  fully  life  history  of  these  orgaisms  has  not  yet  been  worked  out.  What  has 
been  described  under  the  name  Treponema  (Spiroch&ta)  pallidum  is  in  all  probability 
the  male  generation  of  this  organism,  the  female  cells  being  irregular  in  outline  and 
found  in  the  lymphocytes  and  in  other  body  cells. 


318  PHARMACEUTICAL  BACTERIOLOGY 

4.  Spirochata    vincenti. — Pathogenic;  causes    throat    inflammation 
(Vincent's  angina). 

5.  Treponema   pallidum. — The    specific    cause    of    syphilis.     Often 
other  related  organisms  are  found  associated  with  it.     This  organism 
stains  with  difficulty. 

6.  Trypanosoma  Gambiense. — This  is  the  cause  of  the  dread  sleeping 
sickness  of  Africa.     The  transmitter  of  the  infection  is  the  tsetse  fly 
(Glossina  palpalis).    Investigations  have  shown  that  the  destruction 
of  the  tsetse  fly  would  also  eradicate  the  disease  (Koch),  which  has 
practically  depopulated  large  districts  in  Africa.     Related  organisms 
cause  diseases  in  horses  (surra,  dourine  and  mal  de  caderas).     There 
are  also  many  trypanosomes  of  frogs,  fish  and  birds,  but  these  are 
probably  harmless  to  man. 


PJG.  85. — A,  Spirocheta  refringens;  b,  Spirocheta  pallida.      The  cause  of  syphilis. 

III.  FLAGELLATA.     (Mastigophora). — Most  of  the  organisms  belonging 
to  this  group  are  ameboid.     There  may  be  a  fairly  distinct  cell  membrane, 
and  some  have  a  distinct  mouth  part  or  end,  the  so-called  cytostome,  which 
leads  to  a  blind  oesophagus.    They  contain  flagellae,  in  addition  to  pos- 
sessing an  ameboid  movement.     Some  of  the  representatives  of  this  group 
appear  to  have  a  complex  internal  structure.     The  earlier  stages  may  be 
without  flagellae  and  may  be  readily  confused  with  amebae. 

Species  of  Leishmania  cause  sores  and  ulcers  (in  tropical  countries). 
Certain  tropical  Lamblia  and  Trichomonas  species  may  cause  intestinal  and 
other  disturbances. 

IV.  INFUSORIA.    (Cilia ta). — These  have  numerous  very  fine  cilia  and 
contractile  vesicles.     The  bodies  are  oval  and  may  be  free  swimming  or 
attached.     They  have  a  complex  internal  structure  and  are  supposed  to 
be  the  highest  of  the  entire  group  of  protozoa.     There  is  a  distinct  body 
covering  or  membrane  and  a  distinct  mouth  part.    They  are  essentially 
saprophytic  scavengers  being  abundant  wherever  there  is  an  abundance  of 
organic  matter  in  water.     They  are  wholly  aquatic  in  habit. 


PROTOZOA  IN  DISEASE  319 

To  this  group  belong  the  widely  distributed  Paramecia,  which  are  all 
true  scavengers,  feeding  upon  decayed  and  decaying  organic  matter, 
vegetable  as  well  as  animal,  including  the  organisms  which  give  rise~t<T 
the  decomposition  changes.  They  show  marked  preference  for  decay- 
ing vegetable  matter.  For  example,  decaying  fish  food  or  fish  meal 
infusion,  well  diluted  with  water,  is  likely  to  contain  a  pure  culture  of 
Paramecia.  The  accompanying  rotting  bacteria  are  actively  devoured 
by  the  paramecia. 

The  complexity  (physiological  as  well  as  morphological)  of  the  para- 
mecial  cell  has  suggested  the  use  of  this  group  of  organisms  for  the 
purpose  of  making  comparative  toxicological  tests,  in  place  of  the  higher 
animals  now  almost  universally  employed  for  such  tests. 

The  infusoria  proper  (Ciliata),  while  exceedingly  abundant  and  widely 
disseminated,  are  mostly  non-pathogenic.  The  Balantidium  coli  is  a  com- 
mon hog  parasite  which  may  also  cause  serious  dysentery  in  man. 
V.  SPOROZOA. — Have  no  cilia,  move  by  plasmic  contraction  of  the  cell 
and  reproduce  by  spores.  Of  this  group,  the  most  important  species  is  the 
Plasmodium  malaria  which  is  the  primary  cause  of  ague  or  malaria.  The 
carriers  of  the  infection  are  certain  mosquitos  (species  of  Anopheles}. 
If  the  Anopheles  group  of  mosquito  could  be  exterminated  throughout  the 
world,  malaria  would  disappear  also.  The  organism  is  introduced  into  the 
blood  by  the  sting  of  the  insect.  In  the  blood  it  undergoes  certain  cycles 
of  development.  The  fever  paroxysms  are  due  to  the  sporulation  of  the 
organisms  in  the  circulatory  system.  During  the  intervals  (non-sporula- 
tion)  there  is  no  marked  febrile  disturbance.  There  are  several  species  of 
Plasmodium  causing  the  several  forms  of  malaria.  The  tertian  form 
(P.  vivax)  has  a  cycle  of  forty-eight  hours;  the  quartan  (P.  malaria)  has  a 
cycle  of  seventy- two  hours;  and  the  malignant  tertian  (P.  falciparum) 
has  a  cycle  of  forty-eight  hours.  In  the  latter  type  the  paroxysms  are  so 
severe  as  to  give  rise  to  a  continued  fever.  Quinine  is  fatal  to  the  Plasmo- 
dium and  this  remedy  should  be  given  as  a  prophylactic  and  as  a  cure. 

The  draining  of  swamps  and  other  breeding  places  for  mosquitos  has 
reduced  -malaria.  The  use  of  mosquito  netting,  screens,  etc.,  has  also 
checked  this  disease.  Small  water  areas  may  be  treated  with  crude  petro- 
leum oil  which  kills  the  mosquito  larvae. 

The  primary  cause  of  yellow  fever  is  as  yet  unknown  but  it  has  been  def- 
initely determined  that  the  carrier  is  a  mosquito,  the  A'edes  (Stegomyia) 
calopus.  Yellow  fever  is  essentially  a  tropical  disease,  though  it  may 
flourish  in  temperate  zones  until  checked  by  frost  which  is  so  readily  fatal 
to  the  carrier,  the  mosquito.  It  has  been  ascertained  that  the  Aedes  does 
not  occur  far  from  human  habitations,  that  it  breeds  generally  in  barrels 
and  cisterns  containing  rain  water,  rather  than  in  ponds  or  larger  bodies  of 


320  ^  PHARMACEUTICAL  BACTERIOLOGY 

water,  more  remote  from  habitations.  Air  currents  may  carry  them 
greater  distances.  These  discoveries  have  made  possible  a  very  effectual 
campaign  against  this  dread  disease.  The  Federal  Government  aided  by 
State  and  Local  Boards  of  Health  have  insisted  on  a  discontinuance  of 
those  breeding  places  which  can  be  controlled  easily.  The  larger  more 
public  breeding  places  were  covered  with  crude  oil.  Screening  windows 
and  doors  and  sulphur  or  Pyrethrum  fumigation  of  mosquito-infected 
houses  and  rooms  was  insisted  upon  and  individuals  were  instructed  in 
methods  of  self-protection  against  the  bites  of  mosquitos.  As  a  result  the 
yellow  fever  ravages  are  now  reduced  to  a  minimum. 


CHAPTER  XV 

DISINFECTANTS  AND  DISINFECTION.    FOOD  PRESERVATIVES. 

INSECTICIDES1 

The  pharmacist  should  be  well  informed  regarding  disinfectants  and 
their  uses  in  order  that  he  may  assist  physicians  and  health  officers  in  carry- 
ing out  sanitary  rules  and  regulations  in  which  disinfectants  play  50  impor- 
tant a  part.  The  pharmacist  should  know  how  to  disinfect  sick  rooms, 
private  homes  and  public  buildings.  He  should  in  addition  be  informed 
regarding  the  essentials  in  the  construction  of  sanitary  homes,  shops  and 
stores.  He  should  be  able  to  give  good  advice  regarding  water  supply, 
sewage  disposal  and  on  preventive  medicine  generally.  He  should  be  well 
informed  regarding  the  preservation  of  foods,  the  use  and  abuse  of  food 
preservatives  and  on  food  adulteration  and  should  be  prepared  to  test 
foods  as  well  as  drugs  as  to  quality  and  purity.  He  should  be  informed 
regarding  the  nature  and  use  of  insecticides  and  pest  exterminators 
generally. 

Disinfectant  is  synonymous  with  germicide  and  means  any  substance, 
usually  in  the  form  of  a  liquid  or  gas,  capable  of  destroying  bacteria  and 
their  spores,  more  particularly  the  pathogenic  forms.  A  septic  substance 
is  one  contaminated  or  infected  with  pathogenic  or  otherwise  objectionable 
bacteria.  An  aseptic  substance  is  one  free  from  bacterial  infection  or  con- 
tamination, but  not  necessarily  possessed  of  disinfecting  or  even  preserving 
power.  More  broadly  speaking,  disinfectant  means  any  ponderable  or  im- 
ponderable agent  or  substance,  destructive  to  bacterial  life  and  it  is  in  this 
sense  that  the  term  is  here  used.  Preservatives  may  be  defined  as  mild 
disinfectants;  that  is,  when  used  in  larger  amounts  or  stronger  concentra- 
tion, preservatives  become  disinfectants.  Furthermore,  the  term  pre- 
servative usually  applies  to  substances  added  to  foods  for  the  purpose  of 
preventing  or  retarding  microbic  infection  and  microbic  development. 
It  is,  however,  also  applied  to  other  substances.  We  speak,  for  example,  of 
wood  preservatives,  leather  preservatives,  fur  preservatives,  etc.,  meaning 
thereby  substances  which  will  prevent  certain  decomposition  or  other  de- 
tructive  changes  in  the  articles  named,  due  to  a  variety  of  organisms  as 
mould,  larvae,  insects,  mites,  etc. 

1  Each  student  is  required  to  use  the  following  handbook  in  connection  with  the 
study  of  antiseptics  and  their  practical  application.     Dakin  and  Dunham.     Hand- 
book of  Antiseptics.     The  MacMillan  Company,  1917. 
21  321 


322  PHARMACEUTICAL  BACTERIOLOGY 

The  chief  purpose  in  disinfection  is  to  check  and  prevent  the  spread  of 
communicable  diseases,  by  destroying  the  primary  causes  thereof,  namely, 
the  pathogenic  bacteria  or  other  disease  producing  organisms.  The  agents 
or  substances  which  have  disinfecting  powers  or  properties  are  legion. 
We  can  only  refer  to  a  few  of  the  more  important  ones,  those  which  are 
commonly  employed,  giving  the  methods  of  their  use  and  explaining  their 
action. 

Disinfectants  differ  greatly  as  to  germ  destroying  powers  and  attempts 
have  been  made  from  time  to  time  to  standardize  them  or,  in  other  words, 
to  determine  their  comparative  germicidal  efficiency,  but  thus  far  no  satis- 
factory or  generally  acceptable  method  has  been  devised.  All  methods 
appear  to  have  some  objectionable  features.  The  technic  and  principles 
involved  in  the  standardization  of  antiseptics  include  the  following: 

1.  Selecting  some  antiseptic  as  the  unit  of  comparison,  as  a  solution  of 
phenol. 

2.  As  test  objects,  definite  quantities  of  bacterial  cultures  are  used;  as 
bouillon  cultures  of  the  typhoid  bacillus,  colon  bacillus,  hay  bacillus,  etc. 
Some  experimenters  first  air  dry  the  bacteria  before  exposing  them  to  the 
disinfectants  to  be  tested.     There  are  a  number  of  methods  known  as.  the 
"silk-thread  method,"  the  " garnet  method,"  "the  glass-rod  method," 
"the  platinum-loop  method,"  "the  spoon  method,"  and  others. 

3.  Exposing  the  bacteria  from  a  standard  culture,  for  definite  periods 
(uniform  for  the  series  of  tests)  of  time,  to  varying  strengths  of  the  disinfec- 
tants to  be  tested. 

4.  Plating  out  (in  Petri  dishes)  the  exposed  bacteria  in  order  that  the 
death  point  may  be  ascertained. 

The  results  are  expressed  numerically  by  dividing  the  strength  of  the 
disinfectant  tested  which  will  kill  a  given  organism  in  a  given  time  by  the 
strength  of  the  phenol  solution  which  under  the  same  conditions  will  kill 
the  same  organism  in  the  same  time.  To  illustrate,  we  will  suppose  that  a 
1-40  solution  of  formaldehyde  will  kill  the  typhoid  bacillus  in  ten  minutes 
at  37°  C.  and  that  a  i-no  solution  of  phenol  will  kill  the  same  organism  in 
the  same  length  of  time  and  at  the  same  temperature,  then  we  get  as  the 
phenol  coefficient  of  formaldehyde,  0.36  (4%io  =o-36),  which  means  that 
formalin  is  only  about  one-third  as  active  as  phenol  as  far  as  the  destruc- 
tion of  the  typhoid  bacillus  is  concerned.  The  phenol  coefficient1  is 
also  known  as  the  Rideal- Walker  (R-W)  coefficient,  named  after  the  Eng- 

1  For  a  full  discussion  of  the  methods  for  determining  the  phenol  coefficient  and 
the  albumen  coagulation  coefficient,  the  student  is  referred  to  the  following  books: 
Schneider.  Bacteriological  Methods  in  Food  and  Drug  Laboratories.  P.  Blakiston's 
Son  and  Co.,  1915;  or,  Tanner.  Bacteriology  and  Mycology  of  Foods,  John  Wiley 
and  Sons,  IQIQ. 


DISINFECTANTS    AND    DISINFECTION  323 

lish  investigators  who  worked  out  the  details  of  the  method.  In  time  no 
doubt  an  international  standard  method  for  testing  disinfectants  will  be 
adopted.  This  would  be  of  inestimable  value  for  comparative  purposes. 

i.  Physical  and  Mechanical  Disinfectants 

The  following  is  an  outline  of  the  physical  and  mechanical  means  of 
disinfecting. 

a.  Cleanliness. — That  is,  bacterial  cleanliness,  or  absence  of  bacteria, 
brought  about  in  a  variety  of  ways.     The  liberal  use  of  pure  water  for 
washing,  bathing  and  cleansing  purposes,  is  one  of  the  oldest  methods  for 
getting  rid  of  pathogenic  and  otherwise  objectionable  organisms.     It  is,  at 
the  present  time,  one  of  the  most  effectual  means  of  disinfection,  practised 
by  the  housewife,  the  nurse,  the  physician,  in  fact  by  all  classes  and  condi- 
tions of  peoples.     By  bacterial  cleanliness  we  bring  about  a  dilution,  an 
attenuation,  a  dissemination  of  objectionable  organisms  to  such  a  degree, 
that  bacterial  localization  and  infection  are  greatly  retarded  or  are  made 
impossible.     Cleanliness  prevents  filth  and  dirt  accumulation. 

b.  Pure  Air. — Pure  air,  that  is  air  free  from  disease  organisms,  is  a 
prime  essential  in  preventive  medicine.     Not  only  should  the  air  we 
breathe,  be  free  from  bacterial  infection,  but  it  should  also  be  free  from 
smoke,  fumes,  noxious  gases,  soot  and  dust.     The  air  in  many  of  our  large 
cities  is  often  quite  unsuitable  for  breathing  purposes  due  to  fumes,  soot 
and  smoke  from  numerous  furnaces  and  factories,  stenches  from  sewage, 
from  stock  yards,  from  gas  factories,  etc.     This  should  not  be.     Stock 
yards,  glue  factories,  etc.,  should  be  sufficiently  remote  from  cities  to  avoid 
permeating  the  city  with  the  horrible  stenches  emanating  therefrom. 
Smoke,  fumes  and  noxious  gases  should  not  be  permitted  to  escape.     The 
recent  tests  with  smoke  consumers,  with  the  precipitation  of  fumes  and 
smoke  by  means  of  electricity,  etc.,  would  indicate  that  it  is  possible  to 
prevent  the  pollution  of  the  atmosphere  by  the  above  agents.    Just  as  soon 
as  there  is  a  smoke  consumer  on  the  market  that  is  a  practical  success, 
every  smoke  producing  furnace  should  be  supplied  with  one,  irrespective 
of  cost.     The  same  should  apply  to  the  use  of  smelter  fume  precepitators. 
Streets  should  be  kept  comparatively  free  from  dirt  and  dust  by  means  of 
sprinkling  cart  and  street  sweepers  and  cleaners. 

The  "no  spitting"  ordinances  are  largely  a  failure  simply  because  no 
provision  is  made  to  supply  the  appurtenances  necessary  to  carry  them  out. 
It  is  not  sufficient  to  simply  put  up  a  notice  stating  that  "It  is  unlawful  to 
spit  upon  the  floor,"  but  cuspidors,  or  other  receptacles  must  be  provided 
in  sufficient  numbers,  conveniently  placed,  and  furthermore  said  recep- 
tacles must  be  kept  clean  and  sterilized  from  time  to  time,  otherwise  they 
may  become  the  breeding  places  and  disseminators  of  disease.  The 


324  PHARMACEUTICAL  BACTERIOLOGY 

spitting  habit  of  the  adult  male  is  largely  due  to  the  use  of  tobacco,  espe- 
cially chewing  tobacco.  While  it  is  not  possible  to  discontinue  spitting 
altogether,  it  can  certainly  be  greatly  reduced.  Women  rarely  spit  in 
public  and  men  can,  if  they  will,  also  discontinue  the  nasty  habit. 

A  most  serious  defect  in  places  of  habitation  is  the  lack  of  pure  air,  as  in 
small  bed-rooms,  in  the  Pullman  sleepers,  in  sweat  shops,  in  factories,  in 
school-rooms.  Next  to  the  crowded  sweat  shops  in  our  large  cities,  the 
lower  berth  in  the  American  Pullman  car,  is  most  unsuitable  for  human 
habitation.  Rooms  for  living  purposes,  sleeping  purposes,  for  factory  use, 
office  use,  etc.,  etc.,  should  not  only  be  large  enough,  but  there  should 
be  adequate  provision  to  renew  the  air  constantly,  no  matter  how  warm  or 
how  cold  it  may  be.  We  need  a  thorough  sanitary  supervision  of  all 
building  construction,  whether  private  home,  school,  factory,  sleeping  car, 
office,  or  street  car.  There  is  plenty  of  pure  air  and  every  individual 
should  have  an  ample  supply,  for  pure  air  is  one  of  the  most  potent  factors 
in  preventive  medicine. 

c.  Heat. — Heat  is  one  of  the  best  disinfectants  known.    Dry  and  moist 
heat  are  used,  both  of  which  have  been  sufficiently  treated  in  the  preceding 
chapters.     Mere  dryness  is  in  itself  a  germ  destroyer.     Microbes  require 
moisture  for  their  growth.     Most  bacteria  (vegetative  cells,  not  spores)  suc- 
cumb in  a  dry  atmosphere  in  a  comparatively  short  time,  several  hours  to 
several  days.    The  spores  may,  however,  survive  dryness  for  many  months. 

The  dry-air  temperature  usually  employed  for  germicidal  purposes, 
ranges  from  140°  C.  to  170°  C.,  acting  for  one  hour  or  longer.  A  dry  heat 
of  145°  C.  acting  for  one  hour  is  sufficient  to  kill  all  bacteria,  including  the 
spores.  Temperatures  used  for  purposes  of  disinfection  and  sterilization 
range  from  55°  G.  to  120°  C.  55°  to  75°  C.  is  usually  employed  in  the  pasteuri- 
zation of  milk  and  in  sterilizing  sera,  vaccines,  certain  culture  media  (as 
egg  albumen,  blood  serum),  etc.  Moist  heat  of  100°  C.  in  the  form  of  cir- 
culating steam  vapor  is  much  used.  To  obtain  a  moist  temperature  above 
100°  C.,  an  autoclave  is  necessary,  or  liquids  may  be  employed  which  boil 
at  a  temperature  higher  than  100°  C.  as  cumene,  oils,  etc. 

d.  Cold. — Cold,  10°  C.  and  lower,  has  decided  antiseptic  properties,  that 
is,  it  checks  bacterial  growth  and  activity  very  effectually,  as  has  already 
been  explained.    Prolonged  freezing  is,  however,  necessary  to  kill  bacteria. 
Cold  may  therefore  be  considered  a  most  excellent  check  upon  bacterial 
activity,  but  it  is  a  very  poor  germicide.     Cold  is  a  universally  recognized 
and  an  extensively  used  food  preservative,  due  to  its  checking  influence 
upon  bacterial  growth. 

e.  Agitation. — The  agitation  of  gases  and  liquids  reduces  the  bacterial 
activity  therein.     Still  waters  become  stagnant  but  running  waters  do  not, 
in  the  comparative  sense,  due  in  part  to  the  difference  in  the  oxygen  con- 


DISINFECTANTS   AND   DISINFECTION  325 

tent.    Agitating  and  churning  contaminated  liquids  checks  bacterial 
development  somewhat.    The  active  circulation  of  contaminated  air 
reduces  the  number  of  bacteria  present.    Agitation  is,  however,  not.  a, 
satisfactory  means  of  sterilization  and  disinfection. 

/.  Sedimentation  and  Filtration. — Sedimentation  in  sewage  waters  and 
other  contaminated  liquids,  combined  with  nitration,  is  a  very  effective 
means  of  purification.  Precipitation  and  filtration,  aided  by  chemicals  as 
alum,  iron  sulphate,  and  other  coagulants,  are  much  employed  in  the  puri- 
fication of  water  supplies. 

g.  Free  Circulation. — Free  circulation  of  air  and  water  are  most  favor- 
able to  sanitation  because  of  the  checking  influence  upon  bacterial  activity 
and  also  because  of  the  disseminating  and  diluting  effects  upon  the  organ- 
isms which  may  be  present.  Circulation  is  strictly  speaking  a  form  of 
cleansing. 

Purification  of  flowing  water,  as  rivers  and  small  streams,  is  effected 
very  largely  by  oxidation  and  dilution.  The  agitated  water  takes  up 
oxygen  by  absorption  which  combines  with  the  organic  particles  suspended 
in  the  water  rendering  it  unsuitable  as  food  for  bacteria.  As  the  water 
flows  along,  the  bacteria  are  scattered  more  and  more.  Sedimentation 
is  also  an  important  factor  in  the  destruction  of  bacteria.  Gradually 
the  bacteria  settle  to  the  bottom  of  the  stream  where  they  are  brought 
in  competition  with  other  bacteria,  protozoa,  algae,  perhaps  hyphal 
fungi,  etc.,  which  tend  to  check  and  even  entirely  inhibit  their  further 
development. 

h.  Light. — Sunlight  has  most  marked  germicidal  powers,  due  in  part, 
to  the  drying  effects  produced  and  in  part  to  the  actinic  or  chemically 
active  rays  of  the  sun's  light.  Numerous  investigators  have  demonstrated 
the  germ-destroying  effects  of  the  blue  and  violet-rays  and  the  ultra  violet 
end  of  the  solar  spectrum.  Bacteria  cannot  survive  in  sunlight.  Electric 
light  is  said  to  have  the  same  effect  upon  bacterial  h'fe  as  sunlight.  The 
J\T-rays  destroy  bacteria,  likewise  does  radium,  and  these  agents  have  been 
extensively  tested  in  the  treatment  of  skin  diseases  and  superficial  tubercu- 
losis as  lupus,  and  in  cancer,  but  without  satisfactory  or  conclusive  results. 

i.  Electricity. — The  electrical  current  in  itself  appears  to  be  without 
germicidal  powers,  but  electricity  is  used  to  precipitate  smelter  fumes, 
and  organic  impurities  in  water,  as  already  stated.  Electricity  is  used 
to  stimulate  seed  germination  and  it  may  be  possible  to  utilize  electrical 
discharges  or  currents  in  the  treatment  of  communicable  diseases. 

2.  Chemical  Disinfectants 

Chemical  disinfectants  may  be  divided  into  gaseous  (or  vaporous)  and 
liquid  (solutions).  The  liquid  disinfectants  are  superior  to  the  gaseous 
disinfectants  because  direct  contact  with  the  articles  to  be  disinfected  can 


326  PHARMACEUTICAL  BACTERIOLOGY 

be  brought  about,  as  in  washing,  immersing  or  mixing.  Gaseous  disin- 
fectants are  effective  for  surface  sterilization,  especially  useful  for  inac- 
cessible rooms,  buildings,  ships,  paintings,  books,  fabric,  etc.  Both  have 
their  special  advantages,  however. 

The  number  of  chemical  disinfectants,  variously  classed  as  gaseous, 
liquid,  patent,  proprietary,  efficient,  useful,  useless,  etc.,  is  very  great. 
We  shall  mention  only  a  few  of  the  more  powerful  kinds.  No  reliance 
should  be  placed  in  any  patented  or  proprietary  disinfectant  until 
its  value  has  been  demonstrated  by  tests  made  by  reliable  bacteriologists, 
giving  its  phenol  coefficient.  Nor  is  this  all,  not  only  must  the  disinfectant 
have  actual  germ- destroying  powers,  but  it  must  also  be  practically  usable 
and  it  must  not  be  misrepresented  as  to  its  value  and  its  application  and 
use  in  practice. 

The  resistance  of  pathogenic  germs  to  disinfectants  is  extremely 
variable.  Furthermore,  the  various  disinfectants  produce  changes  in  the 
tissues  and  substances  in  and  upon  which  they  act,  which  changes  tend  to 
modify,  check  or  inhibit  the  disinfecting  powers.  Thus  a  number  of 
disinfectants  may  have  the  same  laboratory  phenol  coefficient  and  yet 
their  value  as  disinfectants  in  actual  practice  is  widely  different  because 
of  the  difference  in  the  effects  produced  in  and  upon  the  substances  with 
which  they  are  brought  in  contact. 

As  a  rule,  the  action  and  use  of  disinfectants  is  variable  according  to 
the  following  conditions: 

1.  Disinfectants  are  more  active  when  warm  or  hot.     In  all  disinfec- 
tions hot  solutions  should  be  used,  if  possible  and  if  practicable. 

2.  Gaseous  disinfectants  act  only  in  the  presence  of  moisture,  as  will  be 
explained  under  formalin  and  sulphur  disinfection. 

3.  The  thoroughness  of  disinfection  is  directly  proportional  to  the 
time  that  the  disinfectants  are  allowed  to  act. 

4.  The  activity  of  disinfectants  is  directly  proportional  to  the  degree  of 
concentration,  though  there  are  noteworthy  exceptions.     Absolute  alcohol, 
for  example,  is  of  very  little  value  as  a  disinfectant,  whereas  the  weaker 
solutions  (40  to  70  per  cent.)  are    very  active  germ  destroyers.     The 
same  is  true  of  ether,  chloroform,  glycerin  and  a  number  of  other  sub- 
stances.    Most  disinfectants  have  a  concentration  of  optimum  or  maxi- 
mum efficiency  which  is  the  degree  of  concentration  generally  employed 
in  practice. 

5.  In  actual  practice  the  cost  of  disinfectants  is  a  factor  of  some 
importance,  as  is  indicated  by  the  table  giving  the  comparative  phenol 
coefficient  and  the  relative  cost: 

6.  It  is  known  that  the  disinfecting  power  of  metallic  salts  is  propor- 
tionate to  their  electric  dissociation,  that  is,  the  more  strongly  a  salt  is 


DISINFECTANTS   AND   DISINFECTION  327 

dissociated  by  electrolysis  the  stronger  is  its  disinfecting  power.  It 
follows  that  anything  which  interferes  with  the  electrolytical  dissociation 
of  germicides  weakens  the  germicidal  power.  For  example,  the  addition  of 
sodium  chloride  lowers  the  germ  destroying  powers  of  corrosive  sublimate 
through  such  interference.  This  is  a  matter  of  great  importance  in 
determining  the  efficiency  value  of  antiseptics. 

7.  The  chemical  composition  of  the  material  associated  with  the  germs 
to  be  destroyed,  has  a  marked  influence  upon  the  action  of  the  germicides. 
Thus  germicides  give  different  results  when  acting  upon  the  same  organism 
in  water,  in  beef  broth,  in  salt  solutions,  in  and  upon  tissues,  etc.     For 
this  reason  the  value  of  germicides  in  actual  practice  cannot  be  based 
exactly  upon  uniform  laboratory  results. 

8.  Not  only  do  different  species  of  disease  germs  differ  in  resistance  to 
germicides,  but  the  different  strains  of  the  same  species  react  differently 
with  the  same  germicide.     Certain  substances  appear  to  have  an  elective 
affinity  for  certain  organisms,  as  for  example,  quinine  for  the  malaria  germ, 
and  mercury  salts  for  the  syphilis  germ. 

Disinfectants  destroy  or  kill  germs  in  different  ways.  In  some  cases 
the  death  of  the  organism  is  due  to  oxidation  as  when  ozone,  hydrogen 
peroxide  and  sulphites  are  used,  or  death  may  be  due  to  interference  with 
nutrition,  but  more  generally  it  is  due  to  the  coagulation  of  albumen  and 
abstraction  of  water  from  the  cell-plasm,  as  in  the  use  of  dry  heat,  phenol, 
alcohol,  tannic  acid  and  metallic  salts.  As  already  explained  in  another 
chapter,  lysins  act  by  actually  disintegrating  the  bacterial  cells. 

As  a  rule  germicides  are  most  active  when  dissolved  in  water,  though 
some  authorities  declare  that  bichoride  of  mercury,  phenol,  thymol  and 
lysol  are  more  active  when  dissolved  in  50  per  cent,  alcohol.  The  activity 
of  phenol  as  a  germicide  is  greatly  increased  by  the  addition  of  hydrochloric 
acid,  whereas  lime  reduces  its  potency.  Solutions  of  germicides  in  oils 
are  inert  because  oil  does  not  penetrate  the  bacterial  cell;  however,  the  oil 
itself  may  be  fatal  to  bacterial  life,  in  which  case  the  added  germicide  is 
unnecessary.  Chemical  germicides  do,  however,  increase  the  potency  of 
the  volatile  coal-tar  products  as  gasoline,  benzine  and  xylol,  provided  the 
germicides  are  soluble  in  or  readily  miscible  with  these  substances. 

The  following  are  the  more  important  disinfectants  given  approxi- 
mately in  the  order  of  their  usefulness  and  potency. 

a.  Carbolic  Acid  (Phenol). — Very  widely  used,  in  strengths  of  from 
i  to  5  per  cent.  As  a  disinfecting  wash  for  all  manner  of  septic  things,  a 
5  per  cent,  solution  is  commonly  employed.  A  2.5  per  cent,  (also  the  5 
per  cent.)  solution  is  much  used  as  a  disinfectant  for  hands  and  the  skin 
generally  and  for  septic  wound  irrigation.  A  0.5  to  i  per  cent,  solution  is 
used  as  a  mouth  wash  and  gargle.  Phenol  does  not  kill  spores  hence 


328  PHARMACEUTICAL  BACTERIOLOGY 

should  not  be  used  after  anthrax,  tetanus,  malignant  edema,  and  other 
diseases  due  to  spore  bearing  bacteria.  Phenol  coagulates  albumen,  but 
not  as  actively  as  does  corrosive  sublimate. 

Carbolic  acid  (5  per  cent.)  is  much  used  for  disinfecting  liquid  dis- 
charges in  dysentery,  typhoid,  cholera,  and  for  the  disinfection  of  sputa 
and  expectorations  in  tuberculosis,  in  pneumonia,  etc.,  using  about  two 
times  as  much  of  the  disinfectant  as  material  to  be  disinfected,  allowing 
the  mixture  to  stand  for  several  hours  at  least. 

A  5  per  cent,  solution  may  be  prepared  as  follows: 

Carbolic  acid  (95  per  cent.) 6^  oz. 

Water i  gal. 

Shake  thoroughly  until  all  of  the  acid  is  dissolved. 

Carbolic  acid  does  not  destroy,  bleach  or  discolor  cloth  fabric,  does 
not  corrode  metal,  has  a  marked  characteristic  odor,  is  a  powerful  escha- 
rotic  poison,  and  the  crystals  are  readily  liquefied  by  heat,  by  alcohol 
and  by  water. 

b.  Liquor  Cresolis  Compositus  U.  S.  P. — This  most  efficient  germicide 
is  a  liquid  soap  with  50  per  cent,  cresol,  miscible  in  all  proportions  with 
water.     The  cresols  used  should  have  a  high  boiling-point  (187°  to  i89°C.). 
The  germicidal  power  of  this  substance  is  nearly  double  that  of    carbolic 
acid.     It  does  not  coagulate  albuminous  matter  and  kills  spores. 

There  are  a  number  of  germicides  similar  to  carbolic  acid  having 
marked  germicidal  properties  including  creolin,  cresol  and  lysol.  These 
are  somewhat  superior  to  carbolic  acid.  Lysol  is  a  cresol  mixed  with  soap 
which  greatly  facilitates  the  solution  of  the  cresol,  being  therefore  similar 
to  liq.  cres.  comp.  U.  S.  P.  They  all  kill  spores. 

c.  Tricresol. — Tricresol  is  a  mixture  of  orthocresol,  metacresol  and 
paracresol.     It  dissolves  in  water  in  the  proportion  of  2.5  per  cent,  and  is 
about  three  times  as  active  as  carbolic  acid.     It  is  less  irritating  than 
carbolic  acid  for  which  reason  it  is  preferred  in  sterilizing  sera  (about  0.25 
per  cent.)  and  other  solutions  intended  for  hypodermic  use.     Tricresol 
kills  spores  and  albuminous  matter  does  not  interfere  with  its  action. 

Tricresol,  cresol,  lysol,  solveol,  solutol  and  creolin  are  usually  employed 
(as  germicides)  in  i  per  cent,  solutions  and  are  generally  conceded  to  be 
equal  to  about  2.5  per  cent,  solutions  of  phenol.  They,  however,  have  no 
superiority  over  the  liq.  cres.  comp.  U.  S.  P. 

d.  Formalin. — The  40  per  cent,  commercial  article  is  used.     It  has 
many  advantages  as  a  disinfectant.     It  does  not  injure,  fade  or  decolorize 
cloth  or  other  colored  fabric  and  does  not  corrode  metal  (excepting  hot 
steel  and  iron) .     It  kills  spores  and  is  an  efficient  deodorant.     Albuminous 
matter  does  not  interfere  with  its  action  and  hence  it  is  an  efficient  sick- 
room   disinfectant.     It    disinfects  and  deodorizes   all  discharges  from 


DISINFECTANTS    AND   DISINFECTION  329 

patients   very  quickly  and  completely,  when  used  in  4  to  5  per  cent. 
solutions. 

As  a  gaseous  disinfectant  it  is  active  in  a  moist,  warm  atmosphere.  -It- 
does,  however,  not  kill  insects  and  other  higher  organisms  and  in  this 
regard  it  is  inferior  to  sulphur  dioxide,  but  has  the  advantages  of  not 
decolorizing  fabric  and  being  a  better  deodorant.     There  are  several 
proprietary  disinfectants  composed  of  soap  and  formalin,  as  lysoform. 

e.  Sulphur.  —  Sulphur  in  itself  is  odorless,  tasteless  and  wholly  inert  as 
a  germicide,  but  when  undergoing  oxidation  into  sulphur  dioxide  (com- 
bustion), in  the  presence  of  moisture,  it  is  a  very  active  disinfectant  and 
is  at  the  same  time  fatal  to  insects  and  in  fact  to  all  forms  of  animal  lif  e, 
including  rats,  mice,  etc.  But  it  cannot  be  used  to  disinfect  fine  fabrics, 
paintings,  books,  etc.,  because  of  the  destructive  effects  upon  pigments. 

Under  ordinary  conditions  the  gaseous  substances,  as  formaldehyde 
(formalin)  and  sulphur  dioxide,  are  surface  disinfectants  only  and  are  used 
where  surface  disinfection  is  all  that  is  required,  as  in  the  sterilization  of 
clothing,  wood  work,  walls,  ceilings,  pictures,  furniture,  etc. 

/.  Bichloride  of  Mercury.  —  This  is  the  most  potent  and  most  exten- 
sively used  of  all  antiseptics.  A  i-iooo  aqueous  solution  (used  hot 
whenever  and  wherever  possible)  makes  a  most  satisfactory  germicidal 
wash  for  floors,  walls,  wood  work  of  all  kinds,  in  fact  anything  requiring 
disinfection,  excepting  metals  which  would  be  corroded  (excepting  of 
course  platinum,  gold,  silver)  and  substances  rich  in  albuminous  matter 
as  pus,  sputum,  and  other  sick  room  discharges,  which  are  coagulated  by 
this  germicide,  checking  further  action. 

The  i-iooo  solution  is  sufficiently  powerful  to  kill  all  non-sporogenous 
bacteria  at  the  ordinary  room  temperature  in  one-half  hour.  For  spores  a 
stronger  solution  (1-500)  and  longer  exposure  are  desirable  (one  hour). 

The  chief  disadvantages  to  the  use  of  corrosive  sublimate  are  its 
highly  toxic  nature,  its  corroding  effect  upon  metals  and  it's  coagulating 
effects  upon  albumen  which  hinders  penetration.  It  should  also  be  borne 
in  mind  that  soap  interferes  with  the  action  of  corrosive  sublimate. 

A  i—  1000  solution  is  made  as  follows: 


Bichloride  of  mercury,  6i>£  grs. 

Citric  acid  or  salt,  61%  grs. 

Water,  i      gal. 

The  sodium  chloride  or  citric  acid  is  added  to  retard  the  decomposition 
of  the  bichloride.  Tablets  are  now  on  the  market  made  from  mercury 
cyanide.  They  are  held  to  be  more  decidedly  antiseptic  than  either  the 
iodide  or  the  bichlorid  of  mercury,  and  are  so  prepared  that  one  tablet 
added  to  a  pint  of  water  will  make  a  strength  of  i-iooo. 


330  PHARMACEUTICAL  BACTERIOLOGY 

g.  Chlorinated  Lime. — Also  known  as  chloride  of  lime.  This  is  an 
oxidizing  disinfectant  and  deodorant,  most  extensively  employed  for  the 
disinfection  of  stools,  urine,  sputa  and  other  excreta.  Eight  ounces  of  the 
chlorinated  lime  are  added  to  one  gallon  of  water.  This  solution  is  placed 
in  the  vessel  which  is  to  receive  the  discharges,  using  at  least  double  the 
amount  to  be  disinfected  and  allowing  the  mixture  to  stand  for  one-half 
hour  or  longer.  Chlorinated  lime  destroys  color  and  corrodes  all  textile 
fabrics  and  most  metals.  It  must  be  kept  in  an  air  tight  receptacle  as  it 
loses  in  strength  on  exposure  to  air.  The  solutions  should  be  made  as 
required. 

h.  Lime. — Lime  (unslaked  lime,  quicklime)  is  very  useful  for  the 
destructive  disinfection  of  cadavers  dead  of  infectious  diseases,  using 
twice  the  amount  of  lime,  by  weight,  to  the  substance  to  be  disinfected. 
The  lime  is  powdered  or  crushed  and  packed  about  the  cadaver  in  a  box 
or  coffin.  Neither  water  nor  moisture  need  be  added. 

i  Milk  of  Lime. — Lime  is  slaked  in  the  usual  way.  From  the  slaked 
lime  the  milk  of  lime  is  prepared  by  adding  eight  parts  of  water.  The 
preparation  should  always  be  made  from  freshly  slaked  lime.  It  is  much 
used  for  the  disinfection  of  stools  and  sputum,  using  an  amount  equal  to 
the  amount  of  material  to  be  disinfected.  Whitewash  is  much  used  to 
disinfect  and  preserve  fences,  stables,  sheds,  walls,  ceilings,  etc. 

j.  Copper  Sulphate. — Blue  vitriol  is  a  very  useful  disinfectant  for  sick 
room  excreta  of  all  kinds,  using  a  5  or  10  per  cent,  solution,  bulk  equal  to 
bulk  of  material  to  be  disinfected,  stirring  and  mixing  and  allowing  to 
stand  for  3  to  4  hours.  Iron  sulphate  (copperas)  is  similarly  used,  though 
it  is  somewhat  weaker  in  action. 

k.  Permanganate  of  Potassium. — This  is  another  of  the  oxidizing  anti- 
septics, having  a  rather  limited  use.  It  is  furthermore  comparatively 
expensive.  Freshly  prepared  solutions  are  used,  ranging  in  strength  from 
i-iooo  up  to  5  per  cent.  Quite  extensively  used  as  a  disinfectant  for 
hands.  Has  been  administered  internally  to  oxidize  alkaloidal  /poisons 
in  the  stomach  and  in  the  intestinal  tract. 

The  following  antiseptics  are  used  more  or  less  in  surgery  and  as  skin 
and  other  tissue  disinfectants.  Some  of  them  are  used  as  general  disin- 
fectants, but  as  a  rule  they  are  not  sufficiently  powerful  to  be  of  much 
practical  value. 

a.  lodoform. — Formerly  much  used  as  a  dressing  for  syphilitic  ulcers. 
It  is  not  germicidal  but  has  decided  aseptic  and  sedative  properties,  hence 
also  used  in  scalds  and  burns.  •  It  may,  however,  cause  dermatitis.  It  is 
insoluble  in  water  but  freely  soluble  in  ether  and  alcohol.  The  ointment 
(containing  10  per  cent,  iodoform)  is  still  much  used.  Aristol,  europhen, 
iodol,  losophen  and  nosophen  are  iodoform  derivatives,  have  similar 


DISINFECTANTS   AND   DISINFECTION  331 

properties,  less  odorous,  less  irritating  and  less  poisonous.    The  persistent 
disagreeable  odor  of  iodoform  is  a  great  objection  to  its  use. 

b.  Boric  A  cid  and  Borax. — Boric  acid  is  a  very  mild  antiseptic  and  hence 
is  of  little  practical  value  as  an  active  germicide  but  it  is  a  good  mild  anti- 
septic.    It  can  be  applied  to  comparatively  aseptic  cuts,  bruises,  wounds, 
etc.,  in  saturated  solution  (aqueous)  or  in  powder.     It  can  be  applied  as  a 
dusting  powder  to  many  conditions  where  a  mild  antiseptic  is  indicated. 
In  saturated  solution  it  makes  a  good  gargle,  mouth  wash,  eye  wash,  etc. 

Borax  is  similarly  used  and  has  similar  properties.  The  choice  between 
the  two  is  decided  by  the  difference  in  reaction.  Boric  acid  is  slightly  acid 
in  reaction,  whereas  borax  is  slightly  alkaline.  The  preparation  boro- 
glycerm  is  much  used  as  a  dressing  .for  inflamed  and  infected  mucous 
membranes. 

Sixteen  grains  of  salicylic  acid  and  96  grains  of  boric  acid  dissolved  in  a 
pint  of  sterile  water  makes  Thiersh's  fluid.  This  is  useful  in  cleansing 
mucous  membranes,  such  as  those  of  the  mouth,  nose  and  eye,  and  it 
may  be  used  in  the  form  of  irrigations  for  cleansing  purposes. 

c.  Creosote. — This  excellent  germicide  is  rarely  used  for  general  ex- 
ternal disinfection  though  it  is  more  active  than  phenol  and  does  not  coagu- 
late albumen  and  is  less  toxic  and  less  irritating.     In  doses  of  from  i  to  10 
minims  (given  internally)  it  is  much  used  as  an  antiseptic  and  stimulant 
in  tuberculosis  and  to  correct  intestinal  fermentation.     The  carbonate  of 
creosote  is  said  to  be  especially  efficacious  in  lung  troubles  (tuberculosis). 
Creosote  is  essentially  an  intestinal  antiseptic. 

d.  Hydrogen  Dioxide. — This  is  the  most  active  of  the  oxidizing  dis- 
infectants, used  in  solutions  of  from  10  to  15  per  cent.     It  is  a  very  active 
bleaching  and  deodorizing  agent.     It  is  not  used  for  general  disinfection 
but  is  one  of  the  best  known  local  germicides,  applied  to  abscesses,  ulcers, 
used  as  a  spray,  as  a  gargle,  etc.     Much  employed  in  dental  work.     Used 
by  bacteriologists  to  determine  the  amount  of  bacteria  in  milk  (indicated 
by  gas  liberation  when  added  to  the  milk  in  fermentation  tubes). 

e.  Naphthalene  Derivatives. — These  are  used  as  intestinal  antiseptics 
but  are  of  doubtful  value  in  the  treatment  of  intestinal  diseases.     They  are 
not  acted  upon  in  the  stomach  secretions  but  on  reaching  the  intestinal 
tract  they  undergo  a  chemical  change  and  act  as  antiseptics.     Their  pro- 
longed use  produces  irritation  of  intestines,  bladder  and  kidneys.     To  this 
group  belong  betanaphthol,  betol,  naphthol,  naphthalin,  and  others. 

To  the  group  of  so-called  intestinal  antiseptics  belong  antipyrin,  acetan- 
ilid,  phenacetin,  phenecol,  quinine,  salicylic  acid,  salol,  salophen,  guaiacol, 
resorcin  and  many  other  substances.  Their  value  as  intestinal  antiseptics 
is  very  problematical  and  doubtful. 

/.  So-called  Respiratory  Antiseptics. — There  are    a    great  variety  of 


332 


PHARMACEUTICAL  BACTERIOLOGY 


volatile  or  gaseous  substances  which  are  said  to  act  as  antiseptics  to  the 
respiratory  tract  when  inhaled,  as  oil  of  thyme,  eucalyptol,  oil  of  eucalyp- 
tus, menthol,  camphor,  euthymol,  campho-phenique,  mint  oil,  etc.,  but 
their  value  in  this  regard  is  nil.  They  may  have  some  stimulating 
effect  upon  the  tissues  of  the  respiratory  tract  but  they  do  not  destroy  any 
germs  which  may  be  present  upon  or  within  the  cells  of  the  respiratory 
passages. 

The  following  table  taken  from  the  work  by  Ellis  gives  the  minimum 
proportion  of  germicidal  activity  of  well-known  disinfectants.  The 
figures  indicate  the  strength  of  solution  necessary  to  prevent  bacterial 
development  when  added  to  substances  capable  of  giving  rise  to  bacterial 
growth.  The  figures  are  not  absolute  for  reasons  which  have  been  fully 
set  forth  in  the  beginning  of  this  chapter.  The  table  is  merely  a  guide  to 
the  relative  activity  of  the  germicides  named. 


1.  Very  active  antiseptics. 

Mercuric  iodide, 
Silver  iodide, 
Mercuric  chloride, 
Silver  nitrate, 

2.  Active  antiseptics. 

Osmic  acid, 

Chromic  acid, 

Chlorine, 

Iodine, 

Chloride  of  gold, 

Bichloride  of  platinum, 

Hydrocyanic  acid, 

Bromine, 

Copper  chloride, 

Thymol, 

Copper  sulphate, 

Salicylic  acid, 

3.  Fair  antiseptics. 

Potassium  bichromate, 

Potassium  cyanide, 

Ammonia, 

Zinc  chloride, 

Mineral  acids, 

Lead  chloride, 

Nitrate  of  cobalt, 

Carbolic  acid, 

Potassium  permanganate, 

Lead  nitrate, 

Alum, 

Tannin, 


1-40000 
1-33000 
1-14300 
1-12500 

1-6666 
1-5000 
i  4000 
1-4000 
1-4000 

1-3333 
1-2500 
1-1666 
1-1428 
1-1340 
i-ini 

I-IOOO 


1-909 

1-909 

-714 

-526 

-500 
-500 
-500 

-333 
-285 
-277 

-222 
-207 


DISINFECTANTS   AND   DISINFECTION  333 


4.  Indifferent  antiseptics. 

Arsenious  acid, 
Boric  acid, 
Arsenite  of  soda, 
Hydrate  of  chloral, 
Salicylate  of  soda, 
Iron  sulphate, 
Caustic  acid, 


-166 

-143 
-in 
-107 

-IOO 

-90 
-56 


5.  Feeble  antiseptics. 

Calcium  chloride,  1-25 

Sodium  borate,  1-14 

Alcohol,  i-io 

6.  Very  feeble  antiseptics. 

Ammonium  chloride,  1-9 

Potassium  iodide,  1-7 

Sodium  chloride,  1-6 

Glycerin,  1-4 

Ammonium  sulphate,  1-4 

The  following  table  gives  the  efficiency  value  of  some  disinfectants. 
It  will  be  seen  that  this  value  is  of  necessity  variable,  depending  upon  the 
variation  in  the  market  price  of  the  disinfectants.  In  will  also  be  seen 
that  in  the  proposed  rating  the  remarkably  high  coagulation  coefficient  of 
some  of  the  more  important  chemical  disinfectants  lowers  the  efficiency 
value  greatly. 

The  efficiency  value  of  any  disinfectant  is  found  by  dividing  the  phenol 
coefficient  by  the  other  coefficients  as  follows: 

Phenol    coefficient Efficiency 

Tox.  coefficient  -f-  Coag.    coefficient  +  Comp.  cost          value 

In  the  table  the  first  figure  in  the  comparative  cost  column  (4th  column)  is 
the  market  price  per  pound  of  the  disinfectant  and  the  second  figure  is  the 
comparative  cost  (compared  with  phenol  at  15  cents  per  pound).1 

1  The  comparative  cost  is  a  variable  quantity.  The  present  cost  of  phenol  is  much 
higher  than  15  cents  per  pound.  The  above  figures  would  have  to  be  revised  to  make 
them  applicable  to  the  prevailing  market  price  for  the  several  disinfectants. 


334  PHARMACEUTICAL  BACTERIOLOGY 

EFFICIENCY  VALUE  OF  A  FEW  DISINFECTANTS 


Name  of  disinfectant 

Phenol 
coeff. 

Tox. 
coeff. 

Coag. 
coeff. 

Comp. 
cost 

Eff. 
value 

Special  properties 

Phenol 

I    OO 

I    OO 

I    OO 

0.15 

I    OO 

I    OO 

Odor 

Boracic  acid  

O.  23 

O    O<C 

o  oo 

0.15 

I    OO 

O   2O 

Odorless 

Chloronaphtholeum 

6  06 

o  16 

o  oo 

0.15 

I    OO 

522 

Odor 

Copper  sulphate  

O    CO 

o  30 

7^0 

O.2O 
1  .20 

o  007 

Odorless.        Slight 
color 

Lysol 

2     12 

O   4^ 

o  oo 

0.65 

49  7 

O    44 

Odorless 

Mercuric  chloride  

A  -2    OO 

ro 

6<JO 

1.27 

8  46 

O.o6 

Odorless. 

Neko 

2O    OO 

O    3O 

o  oo 

0.50 

3-3  -3 

s  66 

Odor 

Potassium  permanganate  

0.85 

0.50 

0.00 

0.25 

1.66 

0.04 

Odorless.    Deodor- 
ant.  Color.    Stains 

Silver  nitrate 

38  oo 

•i    oo 

47  "s 

5-64 

77    60 

o  075; 

Odorless      Stains 

Trikresol  

2    62 

o  oo 

O    OO 

0.40 
2  66 

O    T\ 

Odor. 

THE  WORTH  HALE  TOXICITY  COEFFICIENT 

Worth  Hale  of  the  U.  S.  Public  Health  Service  Hygienic  Laboratory 
has  worked  out  a  method  for  determining  the  comparative  toxicity  of 
disinfectants  of  which  the  following  is  a  brief  summarized  outline. 

Test  Animals. — The  animals  upon  which  the  substance  in  question  is 
to  be  tested  shall  be  white  mice  of  not  less  than  15  nor  more  than  30  grams 
weight. 

Dilutions  and  Dosage. — The  dose  is  to  be  calculated  per  gram  of  body 
weight  and  should  when  diluted  equal  between  0.03  and  0.04  c.c.  per  gram 
weight;  that  is,  0.6  to  0.8  c.c.  for  a  2o-gram  mouse.  The  diluent  is  to  be 
distilled  water  and  primary  dilutions  are  to  be  made  of  such  strength 
that  the  dose  is  easily  measured  with  a  i  c.c.  pipette  graduated  into  hun- 


DISINFECTANTS   AND   DISINFECTION 


335 


dredths.  This  is  most  easily  accomplished  by  the  use  of  the  substance  in 
greater  concentration  than  is  required  to  kill  in  the  above  volume 
doses. 

Administration  of  the  Test  Solutions. — After  the  required  dose  of  the 
diluted  disinfectant  has  been  estimated  it  is  measured  into  a  suitable  dish 
and  is*  then  diluted  further  to  the  required  volume  by  adding  sterile  dis- 
tilled water  in  sufficient  quantity.  A  series  of  mice  are  then  injected 
subcutaneously  with  varying  amounts  of  the  substance  until  the  least 
fatal  dose  (L.F.D.)  is  determined,  the  mice  being  kept  under  observation. 

Time  Limit  of  the  Observation. — After  the  animals  have  been  inoculated 
they  are  kept  under  observation  for  a  period  of  24  hours  unless  death 
results  in  a  shorter  period  of  time. 

Phenol  Comparative  Test. — Mice  of  the  same  lot  are  similarly  injected 
with  pure  phenol  properly  diluted  to  make  the  measurements  of  the  dose 
easy  and  then  further  diluted  in  a  small  dish  to  equal  a  volume  dose  of 
0.03  to  0.04  c.c.  per  gram  of  body  weight  and  the  fatal  dose  determined  as 
above.  This  least  fatal  dose  (L.F.D.)  of  phenol  is  unity  and  the  least  fatal 
dose  of  the  substance  in  question  is  estimated  in  per  cent,  of  this. 

Determining  the  Comparative  Toxicity. — The  phenol  toxicity  of  the  dis- 
infectant tested  is  to  the  toxicity  of  phenol  as  x  is  to  100.  The  example 
given  below  would  be  represented  in  the  following  proportion — 4.5: 
18  ::x  :  100  =  25  per  cent.,  that  is  disinfectant  "A"  is  one-fourth  as 
toxic  as  is  pure  phenol. 


Name  of  disinfectant 

Mouse 
weight 

Dose  per  gram 
body  weight 

Result 

Time, 
H.  m. 

Disinfectant  "A"  

21  is 

O    OOI2 

Survived 

20.64 

0.0016 

Survived 

18.32 

0.0018 

Died 

10:30 

19.05 

O.OO2O 

Died 

*:i5 

Pure  phenol  

18  46 

O  .  OO  3  "? 

Survived 

20.  10 

o  .  0040 

Survived 

19.23 

o  .  0045 

Died 

i:i5 

18.90 

0.0050 

Died 

0:25 

Valuable  information  regarding  the  comparative  toxicity  of  many  of 
the  substances  used  as  disinfectants  may  be  obtained  from  a  study  of  the 
comparative  medicinal  doses.  For  example,  the  doses  of  phenol,  betol, 
resorcinol  and  corrosive  sublimate  are  i  grain,  3  grains,  4  grains  and 
Jio  grain  respectively.  These  doses  are  pract'cally  in  proportion  to  the 
toxicity  of  the  substances  named  and  stating  the  dosage  in  the  terms  of 


336  PHARMACEUTICAL  BACTERIOLOGY 

the  phenol  toxicity  coefficient  as  proposed  by  Hale,  we  would  get  the 
following  results. 

Phenol 100 

Betol 33-3 

Resorcinol 25 

Corrosive  sublimate 3000 

As  a  rule,  however,  the  exact  composition  of  disinfectants  is  either  not 
known  or  is  not  disclosed  by  the  manufacturers  and  in  such  cases  the  only 
thing  to  be  done  in  order  to  ascertain  whether  or  not  the  claims  of  the 
manufacturers  are  correct  is  to  make  tests  as  above  outlined.  However, 
in  cases  where  the  composition  of  the  disinfectant  is  definitely  known, 
whether  a  simple  or  compound  substance,  its  comparative  toxicity  can  be 
determined  by  ascertaining  the  toxicity  of  the  ingredients  and  rating  with 
the  standard,  namely  pure  phenol. 

The  Toxicity  and  Germ  Destroying  Power  of  Some  Disinfectants 

The  values  given  are  obtained  from  various  sources  and  in  some  in- 
stances require  further  verification.  The  table  will  serve  as  a  guide  to  a 
valuation  of  the  disinfectants  for  purposes  of  general  disinfection. 

GERM  DESTROYING  POWER  AND  TOXICITY  OF  DISINFECTANTS 

Name  Destroying       Toxicity 

Phenol  I      Phenol  100 

Alcohol o .  03  o .  05 

Alum 0.64  10.00 

Ammonia 2.40  15.00 

Ammonium  chloride 0.03  10.50 

Ammonium  sulphate 0.015  5  •  °° 

Antozone o.oo 

Arsenious  acid o. 50  5000 . oo 

Arsenite  of  soda o .  33  3000 .  oo 

Bacterol i .  58  45 .  oo 

Benetol i .  23  33 .  oo 

Bichloride  of  platinum 10 .  oo 

Boracic  acid o.  23  5 . oo 

Bromine 5 .  oo 

Cabot's  sulpho-naphthol 3  •  87  1 1 .  oo 

Calcium  chloride o. 08  3 . 50 

Camphor 33  -3° 

Carbolene 1.36  n.oo 

Carbolozone i .  48  6 . 40 

Car-sul 2 .  oo  16 .  oo 

Caustic  acid 0.17  120.00 

Chinosol 0.95  25 .  oo 

Chloride  of  gold 12 . 50 

Chlorine J  12 . 50 


DISINFECTANTS   AND   DISINFECTION  337 

GERM  DESTROYING  POWER  AND    TOXICITY    OF  DISINFECTANTS 

(Continued). 

Name                                                                                                                            Destroying  Toxicity 

Phenol  i      Phenol  100 

Chloro-naphtholeum 6 . 06  16 . 06 

Chromic  acid 15  .00  100.00 

Copper  sulphate 0.50  30 .  oo 

Corrosive  sublimate 43  •  oo  3000 .  oo 

Cre-bol-you —  9 .  oo 

Cremolene 1.26 

Creo-carboline  disinfectant 4 .  oo  30 .  oo 

Creola 0.52  12.80 

Creolin  (Pearson) 3.25  18 .  oo 

Creoleum  (Dusenberry) i.oo  9.00 

Creolol  (Rudish's) i .  24  13 .  oo 

Creo-Sul 15 .  oo 

Creosol  (Saponified) i .  03  6 . 40 

Cresoleum 2 . 90  1 1 .  oo 

Cresylone 56 .  oo 

Crude  carbolic  acid .' 2 . 75  90.00 

Cupric  chloride 4.20  80 .  oo 

Cyllin 1 1 .  oo  40 .  oo 

Dioxygen o .  02 

Electrozone o .  90  — 

Ether o .  50 

Ferrous  sulphate 0.27  40 . oo 

Formacone  liquid o .  04 

Formaldehyde o .  30  75 .  co 

Germol 2.12  16 .  oo 

Glycerin  (sp.  g.  1.25) 0.015  °-S° 

Hycol 12.30  32.00 

Hydrate  of  chloral 0.32  10.00 

Hydrocyanic  acid 7 . 50  10000 .  oo 

Hydrogen  peroxide 6 . 30  2 .  oo 

Hygeno  A 3.56  17.00 

Iodine 12.50  400.00 

Iron  sulphate 0.27  40.00 

Izal 8 .  oo  40 .  oo 

Killitol 0.02 

Kreosota 1.26  5 . 60 

Kreotas i .  10  5 . 60 

Kreso 3.92  22.50 

Kresolig 2.18  56 .  oo 

Kretol 0.92  14.00 

Lead  chloride ; i .  50  200 .  oo 

Lead  nitrate 0.83  300.00 

Liquid  creoleum 2  .  oo 

Liq.  cres.  comp.  U.  S.  P 3 .  oo  56 .  oo 

Lincoln  disinfectant i .  48  1 7 .  oo 

Lisapol 50 .  oo 

Listerine o.oi  o.  20 

Lysol 2.12  45 .  oo 

22 


338  PHARMACEUTICAL  BACTERIOLOGY 


Name  Destroying   _Toxicity 

nol  i 


GERM  DESTROYING  POWER    AND    TOXICITY    OF    DISINFECTANTS 

(Continued) 

Phenol  i      Phenol  160 

Mercuric  chloride 43 .  oo  5000 .  oo 

Mercuric  iodide 120.00  1000.00 

Milkol 11.00 

Mineral  acids 1-1.50  1 20 .  oo 

Naphthalene 2 . 50  7 . 50 

Naphthol  phenoline 6 .40  6 . 40 

Neko - 20.00  30.00 

Nitrate  of  cobalt i .  50 

Noncarbolic  disinfectant 7 . 50 

Osmic  acid 20 .  oo  1,000 .  oo 

Phenaco 15 .  oo  32 .  oo 

Phenol  (pure) i .  oo  100 .  oo 

Phenol  disinfectant o .  61 

Phen.  disin.  &  cleans 7 . 50 

Phenol  liquid  U.  S.  P i .  77  80.00 

Phenol  sodique o .  01  4 . 50 

Phenosote 3.43  19.00 

Phenotas  disinfectant i .  37  9 .  oo 

Pi-ne-ex 10 .  oo 

Pino-lyptol ; 0.27  3 . 20 

Platt's  chlorides o.  01 

Potassium  bichromate 3 .  oo  500 .  oo 

Potassium  cyanide . .     3 .  oo  1500.  oo 

Potassium  iodide o. 02  5 . oo 

Potassium  permanganate o.  85  25 .  oo 

Public  health  dis o. 48 

R.  R.  Roger's  disinfec 3  •  °3  56  •  °° 

Salicylate  of  soda o. 30  5 .  oo 

Salicylic  acid 3  • 20  6  •  5° 

Sanax 0.22  22.00 

Sanitas 0.30 

Saponified  cresol i .  03  6 .  oo 

Silver  nitrate 38.00  300.00 

Sodium  borate o. 04  5 .  oo 

Sodium  chloride o.  02  o.  05 

Sulpho-naphthol 3-8?  15  •  oo 

Tarola 3-i2  16.00 

Trikresol * 2 . 62  90.  oo 

2oth  Cent,  disinfectant o.  13  10.  oo 

Veriform  germicide 0.43  15 .  oo 

Victor  sanitary  fluid ., 13  •  oo 

Wescol  disinfectant : 22 . oo 

Worrel's  disinfectant o .  01 

Zenoleum 2.25  19.00 

Zinc  chloride i . S6  100. oo 

Zodane 0.04 

Zodone  (4) - I-62  8-6° 

Zonol. .                   2.37  lo.oo 


DISINFECTANTS   AND   DISINFECTION  339 

3.  Procedures  for  Disinfection 

A.  Surgical  Disinfection. — a.  The  operating  room  must  be  clean  and 
free    from  pathogenic  and  other  objectionable   organisms.     The   room 
must  therefore  be  disinfected  from  time  to  time,  after  the  methocTof 
procedure  for  any  room  which  may  be  assumed  to  be  infected,  as  will  be 
explained  under  room  and  house  disinfection.     As  to  when,  how  often  or 
how  completely  the  operating  room  is  to  be  .disinfected  that  must  be  left 
to  the  judgment  of  the  surgeon  in  charge. 

b.  Surgeons  should  be  especially  careful  regarding  personal  cleanliness, 
irrespective  of  the  routine  personal  disinfection  and  sterilization  performed 
preparatory  to  an  operation.     They  should  always  be  smooth-shaven  as 
the  beard  is  a  carrier  of  germs. 

c.  On  preparing  for  an  operation  the  surgeon  removes  coat,  cuffs  and 
collar  in  an  ante-room;  rolls  up  shirt  sleeves  and  proceeds  to  wash  and 
scrub  hands  with  tincture  of  green  soap,  then  in  1-1.5  Per  cent,  tinct. 
cres.  comp.  U.  S.  P.  or  lysol,  rinse  in  sterile  water,  dry  with  a  clean  sterile 
towel  and  dip  in  50  to  60  per  cent,  alcohol.     Formalin  and  carbolic  acid 
should  not  be  used  as  hand  disinfectants  (by  the  surgeon)  because  of  the 
benumbing  effects  of  these  chemicals,  causing  a  lessening  in  the  delicacy 
of  touch.     A  i  per  cent,  solution  of  potassium  permanganate  is  recom- 
mended as  a  disinfectant  for  hands.     In  many  hospitals  nothing  more 
than  a  thorough  scrubbing  with  green  soap  is  employed  for  the  hands  of 
surgeons,  with  wholly  satisfactory  results. 

Before  entering  the  operating  room  the  surgeon  and  attendants  don 
sterilized  gowns  with  hoods  covering  head,  hair,  and  face  (beard),  leaving 
only  the  mouth,  nose  and  eyes  free.  The  hands  of  the  attendants  are 
covered  with  sterilized  rubber  gloves. 

d.  The  surgical  instruments  are  washed  and  wiped  dry;  boiled  for 
ten  minutes,  in  water  with  i  per  cent,  soda,  and  laid  in  a  tray  containing 
5  per  cent,  carbolic  acid  solution.     Before  using,  they  are  rinsed  in  boiled 
distilled  water.     Never  sterilize  metallic  instruments  in  corrosive  subli- 
mate, or  in  any  corrosive  disinfectants  of  any  kind.     Only  a  short  expo- 
sure would  suffice  to  dull  the  keen  edge  of  knives,  scalpels,  and  other 
cutting  instruments.     Do  not  sterilize  steel  instruments  in  hot  air  as  high 
temperatures  reduce  the  temper,  and  do  not  sterilize  them  placed  with 
rubber  goods. 

B.  Sick  Room  Disinfection.  —  Disinfection  in  the  sick  room  of  a  patient 
afflicted  with  some  communicable  disease,  may  be  divided  into  disin- 
fection of   dejecta,  urine  and  sputum;  disinfection  of  the  patient;  dis- 
infection of  clothing  and  bedding;  disinfection  of  the  sick  room  itself; 
and  precautionary  disinfection  of  the  attending  physicians,  nurses  and 
attendants.     In  case  of  fatal  termination  of  the  maladv  there  is  included 


34°  PHARMACEUTICAL  BACTERIOLOGY 

disinfection  after  postmortem  and  sterilization  of  the  dead  body.  In  all 
cases,  whether  the  patient  dies  or  recovers,  the  entire  sick  room,  including 
bed,  chairs,  bedding,  etc.,  must  be  thoroughly  disinfected.  The  methods 
of  procedure  may  be  outlined  as  follows: 

a.  Disinfection  of  Excreta. — To  disinfect  dejecta,  urine  and  sputum,  a 
4  per  cent,  solution  of  chloride  of  lime  or  a  20  per  cent,  solution  of  milk  of 
lime  will  be  found  very  efficient,  using  amounts  of  the  disinfectants  equal 
to  the  bulk  of  the  excreta  to  be  disinfected,  mixing  well  and  allowing  to 
stand  for  one  hour.     The  disinfectants  are  first  placed  in  the  veesels 
intended  to  receive  the  excreta,  more  being  added  afterward  if  it  is  thought 
desirable.     If  sputa  and  other  excreta  are  received  upon  napkins  or  other 
cloth,  these  should  be  burnt  at  once,  or  if  that  is  not  convenient  they  may 
be    placed    (entirely   immersed)   in  the  disinfectant.     For  tuberculous 
sputum  the  chloride  of  lime  is  best.     Spit  cups  should  be  kept  two-thirds 
full  of  the  4  per  cent,  soluton.     Paper  spit  cups  are  to  be  destroyed  by 
burning  as  soon  as  possible. 

Sulphate  of  copper  (5  to  10  per  cent,  solution),  or  carbo-hydrochloric 
acid  solution  (5  per  cent,  each  of  phenol  and  hydrochloric  acid)  may  be 
used  in  place  of  the  chloride  of  lime  and  milk  of  lime.  Bichloride  of 
mercury  and  phenol  (without  the  hydrochloric  acid)  are  not  very  satis- 
factory for  disinfecting  excreta  because  of  the  coagulating  effects  upon 
albuminous  matter.  Liquor  cres.  comp.  U.  S.  P.,  lysol  and  tricresol  (2-2.5 
per  cent,  solutions)  may  be  used.  Weak  disinfectants  or  untried  patent 
or  proprietary  disinfectants  should  never  be  used  for  above  purposes. 
For  example,  permanganate  of  potassium,  boric  acid,  borax,  glycothy- 
moline,  borol,  etc.,  would  be  valueless  as  disinfectants  for  excreta. 

b.  Disinfection  of  Patient. — This  includes  cleaning  the  body   surface 
with  soap  and  water,  with  50  to  70  per  cent,  alcohol,  washing  with  i  to 
2.5  per  cent,  solutions  of  phenol,  cres.  comp.,  lysol  or  creolin,  when  so 
ordered  by  the  attending  physician.     Bichloride  of  mercury  (1-2000  to 
i-iooo)  may  be  used  for  skin  disinfection.     A  saturated  solution  of  boric 
acid,  normal  salt  solution  or  a  i-iooo  solution  of  permanganate  of  potas- 
sium may  be  used  as  a  wash  or  irrigation  for  non-infected  wounds  and 
cuts,  etc.,  but  not  for  ulcers,  abscesses,  etc. 

Irritating  disinfectants  should  not,  for  very  obvious  reasons,  be  used. 
In  every  case  the  mode  of  procedure  in  the  disinfection  of  the  patient 
will  be  outlined  by  the  attending  physician. 

Nurses,  attendants  and  physicians  must  observe  the  necessary  pre- 
cautions against  becoming  disseminators  of  the  infection  and  must  resort 
to  certain  methods  of  self  disinfection  after  each  visit  to  the  patient,  as  in 
small-pox,  plague,  diphtheria  and  other  communicable  diseases. 


DISINFECTANTS   AND   DISINFECTION  341 

c.  Disinfection  of  the  Clothing  Worn  by  the  Patient  and  of  the  Bedding. — 
All  clothing  worn  by  the  patient  and  all  bedding,  as  soon  as  ordered 
changed,  should  at  once  be  immersed  in  a  hot,  5  per  cent,  solution  of 
carbolic  acid  or  a  2.5  per  cent,  solution  of  cres.  comp.  or  lysol.     After 
soaking  for  several  hours  the  clothing  should  be  boiled  in  water  for  30 
minutes  at  least.     After  thorough  drying,  perferably  in  the  sun,  the  cloth- 
ing should  be  well  ironed.     The  ironing  process  in  itself  has  very  marked 
germicidal   powers.     Clothing  may  also  be  disinfected  in  formalin  (4 
per  cent.).     Sulphate  of  copper  and  sulphate  of  iron  discolor  and  cor- 
rode the  cloth.     All  cloth  fabrics  and  clothing  which  has  been  in  close 
contact  with  a  patient  suffering  from  diphtheria,  cholera,  plague  or  small- 
pox, should  be  destroyed  by  burning  whenever  feasible. 

d.  Disinfection  of  the  Sick  Room. — The  bed  frame,  the  chairs  and  other 
wooden  furniture,  the  floor  and  the  wood  work  of  the  room,  may  be  washed 
or  wiped  with  corrosive  sublimate  (i-iooo),  formalin  (3-4  per  cent.)  or 
phenol  (5  per  cent.),  if  contamination  is  suspected  or  if  so  ordered  by  the 
physician,  even  while  the  room  is  still  occupied  by  the  patient. 

Just  as  soon  as  the  patient  is  taken  from  the  room,  a  thorough  disinfec- 
tion should  be  carried  out  at  once,  the  disinfection  including  furniture, 
clothing  of  the  patient,  bedding,  mattresses,  pillows,  etc.,  excepting  such 
articles  as  are  ordered  destroyed  by  burning. 

Every  pharmacist  should  fully  inform  himself  regarding  the  state  laws 
and  city  ordinances  governing  health  and  quarantine  regulations.  State 
and  city  boards  of  health  usually  issue  free  bulletins  on  methods  of  disin- 
fection in  communicable  diseases.  Copies  of  these  should  be  on  hand  for 
ready  reference. 

For  room  disinfection,  formalin  or  sulphur  are  used.  With  formalin 
the  procedure  is  as  follows:  For  every  1000  cubic  feet  of  space  there  is. 
required  one  pint  of  formaldehyde  (the  40  per  cent,  commercial  formalin) 
and  8  ounces  of  commercial  potassium  permanganate.  Place  the  perman- 
ganate in  an  agate  lined  or  iron  pail  of  about  ten  times  the  capacity  of 
the  disinfectant  to  be  used,  spreading  the  permanganate  evenly  over  the 
bottom.  Set  pail  containing  the  crystals  upon  a  brick,  iron  stand  or  other 
support,  in  a  tub,  pan  or  dish  partially  filled  with  water.  See  that  win- 
dows and  doors  are  closed  and  sealed  (excepting  the  exit).  The  room 
should  be  warm  and  moist,  a  condition  which  may  be  effected  by  sus- 
pending sheets  wrung  out  of  hot  water  about  the  room.  In  a  steam 
heated  flat,  steam  may  be  allowed  to  escape  from  the  air  vent  of  a  radia- 
tor; or  steam  may  be  generated  outside  of  the  room  and  conducted  in- 
to it  by  means  of  rubber  tubing.  Do  not  have  an  open  fire  or  flame  in 
the  room  to  be  disinfected  as  the  gas  to  be  liberated  is  somewhat  inflam- 
mable. Having  ascertained  that  all  is  in  readiness,  pour  the  formalin 


342  PHARMACEUTICAL  BACTERIOLOGY 

solution  from  a  dipper  or  wide  mouthed  vessel  over  the  permanganate; 
leave  the  room  at  once,  close  and  seal  exit,  plugging  key  hole  and  crevices 
in  door.  Eighty  per  cent,  of  the  gas  is  liberated  within  ten  minutes  or 
less.  Leave  the  room  sealed  for  at  least  six  hours,  preferably  twelve 
hours.  At  the  end  of  this  time  disinfection  is  complete.  Open  doors 
and  windows.  Traces  of  formalin  may  be  destroyed  by  sprinkling  or 
spraying  ammonia  in  the  room. 

•It  is  advised  to  use  a  separate  container  for  every  pint  of  formalin  used. 
A  large'piece  of  matting  or  other  absorptive  material  may  be  placed  under 
-each  container  to  guard  against  the  possibility  of  staining  the  floor,  in 
case  the  floor  requires  such  protection. 

In  case  sulphur  is  used,  prepare  the  room  (as  to  sealing,  air  moisture 
and  warmth)  as  for  formalin  disinfection,  taking  the  precaution  to  remove 
(and  disinfect  separately,  by  means  of  formalin  and  bichloride  of  mercury) 
paintmgs,  clothing  and  other  fabric  which  must  not  be  bleached  by  the 
sulphurous  acid  fumes.  For  every  1000  cubic  feet  of  space,  use  3.5  pounds 
of  flower  of  sulphur.  Place  the  sulphur  on  a  bed  of  sand  or  on  ashes  in 
an  iron  pot  or  pan  which  is  supported  on  a  brick  or  iron  stand  in  a  dish 
of  water.  Pour  a  tittle  alcohol  over  the  sulphur  and  ignite. 

Sulphur  candles  are  now  found  upon  the  market  and  are  more  con- 
venient than  sulphur.  Place  a  sufficient  number  of  the  candles  upon 
bricks  in  pans  of  water  and  light  them.  Liquefied  sulphur  dioxide  put 
up  in  convenient  containers  may  be  employed,  using  15  ounces  to  each 
1000  cubic  feet.  Open  the  can  by  means  of  a  can  opener,  set  it  in  a  pan 
or  dish  and  allow  the  gas  to  evaporate. 

Remember  that  the  sulphur  dioxide  corrodes  metal,  bleaches  clothing, 
hangings  and  draperies  and,  like  formalin,  is  without  disinfecting  power 
in  the  absence  of  moisture. 

After  the  disinfection  with  formalin  or  sulphur  dioxide  is  completed, 
it  is  often  desirable  t6  go  over  the  floors,  furniture,  bed  frames,  etc.,  with 
a  i-iooo  bichloride  of  mercury  solution. 

Mattresses,  heavy  quilts,  pillows  and  furniture  cushions  are  difficult 
to  disinfect  with  formalin  or  sulphur  dioxide.  These  should  be  disin- 
fected by  steam  under  pressure.  In  such  diseases  as  plague,  diphtheria 
and  cholera,  such  articles  should  be  destroyed  by  burning.  Anyway,  a  sick 
room  should  have  simple  furniture  and  merely  such  articles  as  are  ab- 
solutely necessary  and  such  as  can  be  disinfected  readily. 

The  so-called  carbo-gasoline  method  of  book  disinfection  is  highly 
recommended.  Immerse  books,  papers,  clothing  and  other  articles  to  be 
olisinfected  for  twenty  minutes  in  the  carbolized  gasoline.  Take  from 
the  disinfecting  solution  and  allow  to  dry  in  the  open.  The  carbolized 
gasoline^consists  of^Baume  88°  gasoline  or  gas  machine  gasoline  to  which 


.      DISINFECTANTS   AND   DISINFECTION  343 

2  per  cent,  of  carbolic  acid  is  added.  No  injury  is  done  to  the  books  or 
clothing,  provided  they  are  carefully  handled  until  dry.  Gasoline  will, 
however,  injure  oil  paint  lettering,  etc. 

D.  Postmortem     Disinfection    and    Sterilization    of    Cadaver. — After 
autopsies  on  bodies  after  infectious  disease,  thorough  disinfection  must 
be  resorted  to.     A  liberal  use  of  a  4  per  cent,  solution  of  calcic  hypochlor- 
ite,  allowing  this  to  act  for  at  least  one  hour,  will  serve  the  purpose. 

In  cases  of  death  from  contagious  diseases  all  orifices  of  the  body  should 
be  packed  with  cotton  well  soaked  in  a  1-500  bichloride  solution.  The 
entire  body  should  be  washed  with  i-iooo  bichloride  solution.  Crema- 
tion is  desirable  and  the  funeral  should  be  private. 

The  so-called  embalming  fluid  of  funeral  directors  are  aqueous  solu- 
tions of  various  chemical  disinfectants,  having  corrosive  sublimate  and 
formalin  as  the  chief  ingredients.  The  following  formula  is  said  to 
have  the  approval  of  the  National  Funeral  Directors  Association  of 
the  United  States: 

Formalin  (40  per  cent.) n      Ib. 

Glycerin 4     Ib. 

Borax 2 . 5  Ib. 

Boric  acid i      Ib. 

Potassium  nitrate 2 . 5  Ib. 

Solution  of  eosin  (i  per  cent.) i     oz. 

Water,  to  make 10    gal. 

The  salts  are  dissolved  in  six  gallons  of  water;  the  glycerin,  formalin 
and  eosin  added  and  enough  water  to  make  up  the  ten  gallons. 

E .  Disinfection  of  Public  Buildings  and  Public  Conveyances. — Only  rarely 
will  it  become  necessary  to  disinfect  an  entire  large  building,  whether 
private  or  public,  and  then  the  method  of  procedure  is  much  the  same  as 
for  the  sick  room  disinfection,  already  described,  treating  each  room  as 
though  it  were  independent  of  other  rooms,  excepting  that  inner  con- 
necting rooms  need  not  be  closed  and  sealed. 

In  disinfection,  one  important  fact  should  never  be  lost  sight  of,  namely, 
that  it  is  just  as  important  to  destroy  the  carriers  of  disease  (flies,  fleas,  rats, 
mice,  and  other  animals),  as  the  disease  germs  themselves.  This  is  par- 
ticularly important  in  public  disinfection,  so  much  so  that  it  is  a  general 
rule  to  always  use  a  disinfectant  which  destroys  the  disease  carriers,  as  sul- 
phur dioxide.  In  the  yellow  fever  district,  for  example,  the  chief  fumi- 
gating agent  is  burning  Pyre  thrum  which  is  a  sure  death  to  the  Aedes 
mosquito  as  well  as  to  other  insects. 

Wherever  and  whenever  practical  therefore,  sulphur  dioxide  should  be 
used  for  public  disinfection.  In  many  European  cities,  the  health  depart- 
ment is  provided  with  portable  generators  which  are  run  alongside  the 


344  PHARMACEUTICAL  BACTERIOLOGY 

building  to  be  disinfected,  the  sulphur  dioxide  generated  and  conducted 
into  the  room,  hall,  cellar,  or  area  way  to  be  disinfected,  by  means  of  tubing. 
This  is  the  safest  and  most  satisfactory  way.  If  such  apparatus  is  not 
available,  the  flower  of  sulphur,  sulphur  candles,  or  liquefied  sulphur  dioxide 
may  be  used  (15  ounces  to  each  1000  cubic  feet  of  space).  Street  cars, 
railway  cars,  large  public  conveyances  generally,  may  be  disinfected  much 
like  rooms,  after  being  well  sealed.  A  safe  rule  is  to  use  double  quantities 
of  the  disinfectant  for  public  conveyances,  as  compared  with  a  sick 
room,  because  of  the  fact  that  it  is  difficult  to  seal  such  public  con- 
veyances well.  After  the  disinfectant  has  acted  for  a  sufficient  length 
of  time  (twelve  to  twenty-four  hours),  the  place  is  opened,  aired  and  then 
all  of  the  wood  work  (of  furnishings  as  well  as  the  floor,  walls  and  ceiling) 
is  either  washed  or  sprayed  with  a  i-iooo  bichloride  of  mercury  solution 
or  a  3-5  per  cent,  formalin  solution. 

In  such  communicable,  diseases  as  have  no  animal  carriers  (other  than 
the  patient  himself)  or  where  for  obvious  reasons  such  carriers  are  not 
present,  formalin  will  always  be  the  preferred  disinfectant,  whether  for 
private  or  public  disinfection,  bearing  in  mind  that  heat  and  moisture 
are  necessary  adjuncts  to  its  use.  Formaldehyde  is  not  effective  in  a  dry, 
cold  atmosphere  because  under  those  conditions  the  formalin  is  converted 
into  solid  polymerized  paraformaldehyde,  which  as  such,  is  inert. 

Public  or  private  disinfection  by  means  of  formalin  may  be  carried 
out  as  follows,  the  method  selected  depending  upon  time,  place  and 
opportunity. 

a.  Wet  Blanket  Method. — Immerse  blankets  or  sheets  in  the  formalin 
solution  and  suspend  them  about  the  room  to  be  disinfected.     The  room 
may  first  be  sprayed  with  a  hot  4  per  cent,  solution  of  formalin  which 
furnishes   warmth   and   moisture.     The    operator   must   work    rapidly 
as  formalin  is  very  irritating  to  eyes  and  respiratory  tract. 

b.  Methyl  Alcohol  Lamps. — Formalin  may  be  generated  in  the  space  to 
be  disinfected  by  oxidizing  the  methyl  alcohol  and  converting  it  into  for- 
maldehyde.   Lamps  of  special  construction  are  necessary.     The  vapor  of 
methyl  alcohol  is  passed  over  a  highly  heated  plate  whereupon  it  is  oxi- 
dized into  formaldehyde   (CH3OH+O  =  HCHO+H2O)   with  liberation 
of  water.     This  method  of  disinfection  is  now  rarely  employed. 

c.  Sanitary  Construction  Company's  Lamp. — The  mechanism  consists  of 
a  tank   to  hold  the  formalir,   connected  with  a  spiral  tube  through 
which  the  solution  is  slowly  passed  through  a  flame.     The  heat  vaporizes 
the  formalin  which  is  then  conducted  into  the  room  (through  the  key  hole) 
by  means  of  suitable  tubing.     This  apparatus  is  much  used  by  health 
officers. 

d.  The  Shering  Lamp. — These  small  compact  and  most  convenient 


DISINFECTANTS    AND    DISINFECTION  345 

lamps  can  be  secured  from  any  wholesale  drug  supply  house.  With  this 
apparatus  the  solid  tablets  of  paraform  or  paraformaldehyde  are  used. 
The  heat  from  the  lamp  decomposes  the  tablets,  producing  formaldehyde. 
The  lamps  are  placed  in  position,  in  sufficient  numbers,  lighted  and  the~ 
small  tray  of  each  lamp  is  supplied  with  a  sufficient  number  of  tablets. 
As  a  precautionary  measure  each  lamp  should  be  placed  on  a  brick  in  a 
pan  or  dish  of  water.  The  air  in  the  room  must  be  warm  and  moist. 

e.  Formaldehyde  Candles. — These  consist  of  a  mixture  of  paraformalde- 
hyde and  paraffin,  wax,  tallow  or  other  combustible,  which  may  be  moulded 
into  candles.  The  candles  are  placed  in  a  fireproof  dish  or  pan  and  ignited. 
For  room  disinfection  these  candles  are  most  convenient  as  well  as 
satisfactory. 

F.  Disinfection  at  Quarantine  Stations. — All  civilized  nations  maintain 
a  system  of  vigilance  as  a  protection  against  the  introduction,  from  foreign 
countries,  of  certain  communicable  diseases  designated  as  quarantinable. 
The  first  disease  against  which  a  quarantine  was  established  was  the  plague. 
In  the  fourteenth  century  certain  Italian  cities  established  a  quarantine 
against  this  dread  disease  and  the  word  " Quarantine"  came  into  general 
use  because  of  the  fact  that  the  period  of  detention  was  about  forty  days 
(Ital.  quarantind).  The  actual  period  of  detention  as  now  enforced  varies 
somewhat  depending  upon  the  nature  of  the  disease  against  which  the 
detention  is  maintained,  as  determined  by  the  period  of  incubation.  The 
quarantinable  diseases  recognized  by  the  United  States  are  plague  (bu- 
bonic), small-pox,  yellow  fever,  Asiatic  cholera,  leprosy  and  typhus.1 
The  enforcement  of  the  quarantine  regulations  is  under  the  direction 
of  the  U.  S.  Public  Health  Service.  The  most  important  quarantine 
stations  in  the  United  States  are  at  San  Francisco,  New  Orleans,  New  York 
and  Boston,  ranking  in  importance  in  the  order  named.  The  Station 
at  San  Francisco  is  of  special  importance  because  upon  its  efficiency 
depends  very  largely  the  exclusion  of  plague,  cholera  and  small-pox,  the 
three  highly  communicable  diseases  so  prevalent  in  the  Orient.  Of 
course  a  national  quarantine  to  be  effective  must  be  complete,  covering 
every  port  of  entry,  whether  large  or  small,  maritime  or  inland.  This  is 
very  often  not  the  case  and  as  a  result  an  epidemic  may  enter  via  a  minor 
port  where  the  service  is  inadequate  due  to  incompetent  or  insufficient 
inspection. 

The  quarantine  officers  are  kept  informed  as  to  the  occurrence  of  epi- 
demics or  sporadic  cases  of  quarantinable  diseases  in  foreign  countries 

1  National  quarantine  against  foreign  disease  is  entirely  distinct  from  state  or  city 
quarantine.  The  following  diseases  are  recognized  as  quarantinable  by  most  state 
boards  of  health:  Scarlet  fever  (including  scarlatina  and  scarlet  rash),  diphtheria  (in- 
cluding membranous  croup),  small-pox,  epidemic  cerebro-spinal  meningitis,  anterior 
poliomyelitis,  leprosy,  and  bubonic  plague. 


346  PHARMACEUTICAL  BACTERIOLOGY 

and  port  cities  thus  putting  them  on  their  guard  as  to  the  need  of  special 
vigilance  regarding  imports  and  immigration  from  such  places  or  cities. 
However,  every  ship  from  a  foreign  port  on  arriving  within  the  quarantine 
zone  of  the  station  is  visited  by  the  boarding  officer  who  immediately 
proceeds  to  get  data  regarding  the  sanitary  conditions  on  board,  as  to 
deaths,  sickness  of  any  kind,  etc.  All  passengers,  including  the  ship's 
crew,  are  lined  up  and  inspected  by  the  boarding  officer.  If  nothing  unto- 
ward is  reported  or  detected  the  captain  of  the  ship  is  given  a  clean  bill 
of  health  and  the  vessel  is  permitted  to  dock  and  discharge  passengers  and 
cargo. 

If  however  the  boarding  officer  finds  a  case  of  small-pox  or  other  quar- 
antinable  disease  on  board,  the  ship  is  anchored  near  the  station;  the 
passengers  and  crew  are  landed  at  the  quarantine  station  and,  with  the 
aid  of  the  ship's  officers,  the  quarantine  officer  proceeds  to  disinfect  all 
persons  and  their  personal  effects,  the  same  class  distinction  (first  cabin, 
second  cabin,  steerage,  ship's  crew)  being  maintained  as  on  ship.  Each 
day,  as  long  as  the  quarantine  lasts,  all  persons  are  examined  by  the  chief 
officer  of  the  station,  to  note,  if  possible  the  first  manifestations  of  new 
cases.  Just  as  soon  as  a  new  case  is  found  the  patient  is  at  once  taken 
care  of  in  an  isolated  hospital.  Suspects  are  kept  under  observation  in 
an  isolation  camp. 

All  personal  effects,  including  every  bit  of  clothing  worn,  is  disinfected 
in  enormous  double  walled  cylinders,  by  means  of  hot  formalin  laden  steam 
under  pressure.  Sterilization  is  made  absolutely  complete  without  any 
injury  to  the  clothing. 

The  ship  with  its  cargo  is  next  disinfected  with  sulphur  dioxide  gas 
generated  in  iron  pots  or  pails  placed  upon  sheets  of  tin.  A  little  alcohol 
is  poured  over  the  sulphur,  ignited,  the  exits  closed  down  and  kept  closed 
for  twelve  hours.  If  the  cargo  contains  combustible  material  as  alcohol, 
oil,  benzine,  etc.,  the  sulphur  dioxide  is  generated  upon  a  special  boat  or 
float  which  is  run  alongside  and  the  fumes  conducted  into  the  hold  of  the 
ship  to  be  disinfected.  The  sulphur  fumes  kill  all  organisms  present,  in- 
cluding fleas,  rats  and  mice.  In  fact  sulphuring  of  ships  must  be  resorted 
to  [quite  frequently  for  the  sole  purpose  of  killing  rats  and  mice,  even 
though  there  may  have  been  no  disease  on  board. 

4.  Purification  and  Sterilization  of  Water  Supplies 

Every  city,  town,  hamlet  and  home  should  have  an  ample  supply  of 
pure  water  for  drinking,  cooking  and  cleansing  purposes.  Impure  waters, 
that  is  waters  which  require  sterilization  in  order  to  render  them  potable, 
are  always  dangerous.  It  is  therefore  of  prime  importance  to  secure  a  pure 
supply  of  water,  sufficiently  pure  to  make  the  work  of  sterilization  and 


DISINFECTANTS   AND   DISINFECTION  347 

purification  wholly  unnecessary;  if  that  is  not  possible,  and  it  generally  is 
not,  under  our  peculiar  communal  condition,  then  said  questionable  water 
supply  should  be  thoroughly  sterilized  and  purified,  according  to  the  most 
approved  modern  methods.  We  cannot  condemn  too  strongly  the  gen-~ 
erally  prevalent  methods  of  emptying  the  sewage  of  our  cities  and  towns 
into  rivers  and  lakes  and  then  again  supplying  this  sewage  contaminated 
water  to  towns  and  cities  for  drinking  and  cooking  purposes.  There 
should  be  an  efficient  state  board  of  health  cooperating  with  a  federal 
department,  and  there  should  be  efficient  and  competent  sanitary  inspec- 
tors to  look  after  the  water  supplies  of  private  homes,  of  towns  and  in  the 
country. 

The  suitability  of  water  for  drinking  purposes  is  inversely  proportional 
to  the  number  of  bacteria  present.  Pure  spring  or  well  water  contains  very 
few  bacteria,  rarely  exceeding  50  per  cc.  Sewage  contaminated  water, 
which  is  still  used  for  drinking  and  cooking  purposes,  may  contain  several 
million  bacteria  per  cc.  It  has  been  proven  time  and  again  (statistically) 
that  the  mortality  rate  (due  to  disease)  of  cities  is  inversely  proportional 
to  the  purity  of  the  drinking  water  supply.  It  is  self  evident  that  water 
purification  should  be  considered  a  subject  of  the  utmost  importance.  It 
should  receive  more  attention  than  it  does. 

The  sedimentation  and  filtration  method  for  removing  dirt,  sand  and 
other  coarser  particles  from  the  water  supplies  of  large  cities,  is  practised 
and  has  been  practised  for  years  in  many  of  the  European  cities.  This 
is  satisfactory  as  far  as  it  goes,  but  it  does  not  go  far  enough.  The  filter- 
ing material  used  (sand,  charcoal,  etc.)  does  not  remove  bacteria  and  other 
small  organisms,  excepting  those  which  are  attached  to  the  coarser  par- 
ticles remaining  upon  the  filtering  material.  Furthermore,  unless  the 
filter  is  frequently  changed  or  sterilized,  the  filtering  material  will  become 
the  breeding  place  of  germs  and  thus  contaminate  the  water  still  more. 

Various  chemical  disinfectants  have  been  tried,  but  most  of  them  have 
proven  unsatisfactory  for  various  reasons.  The  use  of  high  attenuations 
(1-5,000,000  to  1-50,000)  of  copper  sulphate  has  been  highly  recommended, 
especially  by  the  U.  S.  Dept.  of  Agriculture,  and  has  in  many  instances 
given  excellent  results,  especially  in  the  destruction  of  low  forms  of  algae 
and  protozoa.  As  a  means  of  destroying  bacterial  life  the  method  is, 
however,  not  a  success.  Dr.  Kraemer  and  others  recommend  the  use  of 
copper  foil  or  plates  immersed  in  the  water  as  a  means  of  destroying  patho- 
genic and  other  bacteria,  but  this  method  does  not  appear  to  have  met 
with  any  general  approval.  Kraemer  sums  up  the  copper  foil  treatment 
of  water  as  follows: 

i.  The  intestinal  bacteria,  like  colon  and  typhoid,  are  completely  de- 
stroyed by  placing  clean  copper  foil  in  the  water  containing  them. 


348  PHARMACEUTICAL  BACTERIOLOGY 

2.  The  effects  of  colloidal  copper  and  copper  sulphate  in  the  purifica- 
tion of  drinking  water  are  in  a  quantitative  sense  much  like  those  of  filtra- 
tion, only  the  organisms  are  completely  destroyed. 

3.  Pending  the  introduction  of  the  copper  treatment  of  water  on  a  large 
scale  the  householder  may  avail  himself  of  a  method  for  the  purifications 
of  drinking  water  by  the  use  of  strips  of  copper  foil  about  3  J£  inches  square 
to  each  quart  of  water,  this  being  allowed  to  stand  over  night,  or  from  six 
to  eight  hours,  at  the  ordinary  temperature,  and  then  the  water  drawn  off 
or  the  copper  foil  removed. 

The  alum  method  of  purifying  water  has  met  with  considerable  success, 
but  more  recently  the  alum-sodium  hypochlorite  combination  has  proven 
more  satisfactory.  The  alum  coagulates  and  precipitates  the  organic  im- 
purities and  the  sodium  hypochlorite,  through  its  electric  dissociation,  acts 
as  a  germ  destroyer.  The  coagulated  and  precipitated  organic  material 
holding  most  of  the  bacteria  is  then  removed  by  filtration.  The  amount  of 
chemicals  used  depends  somewhat  upon  the  degree  of  contamination. 
With  highly  contaminated  waters  it  is  customary  to  use  3.3  per  cent,  of 
alum  as  the  coagulant,  subsequently  introducing  1.2  per  cent,  of  the  hypo- 
chlorite. The  water  is  then  filtered,  whereupon  it  is  ready  for  use. 

Small  quantities  of  drinking  water  may  be  purified  as  follows:  Dis- 
solve a  level  teaspoonful  of  powdered  chloride  of  lime  in  a  teacup  of  water. 
This  solution  is  diluted  with  three  cupfuls  of  water,  and  a  teaspoonful  of 
this  mixture  may  be  added  to  each  two-gallon  pail  of  drinking  water. 
This  will  give  0.4  or  0.5  part  of  free  chlorine  to  a  million  parts  of  water  and 
will,  in  ten  minutes,  destroy  all  typhoid  and  colon  bacilli  or  other  dysentery- 
producing  organisms  in  the  water.  •  Moreover,  all  traces  of  chlorine  will 
disappear  rapidly. 

There  are  in  use  a  number  of  methods  for  dissociating  sodium  hypo- 
chlorite by  electricity.  Some  of  them  are  patented  and  modifications 
thereof  are  in  use  by  city  water  purification  works,  giving  excellent  results. 
Dr.  C.  P.  Hoover,  assistant  chemist  of  the  Columbus  Board  of  Health,  has 
the  following  to  say  regarding  the  process: 

''There  are  two  general  types  of  electrolyzers  for  dissociating  sodium 
chloride.  In  one  the  cathodic  and  anodic  products  are  allowed  to  recom- 
bine  in  the  main  body  of  the  electrolyte  and  in  the  other,  known  as  the 
diaphragm  process,  the  products  are  removed  separately  from  the  cell  as 
produced. 

"For  the  production  of  sodium  hypochlorite  the  non-diaphragm  process 
has  been  considered  best  because  it  dispenses  with  the  destructible  dia- 
phragms and  the  loss  of  energy  that  all  such  diaphragms  occasion. 

"When  a  direct  current  of , electricity  is  passed  through  a  solution  of 
sodium  chloride,  sodium  is  liberated  at  one  pole  and  chlorine  at  the  other. 


DISINFECTANTS   AND   DISINFECTION  349 

The  liberated  sodium  reacts  on  the  water  breaking  it  up  into  hydrogen  and 
hydroxyl  ions  to  form  sodium  hydrate.  The  sodium  hydrate  in  turn  com- 
bines with  the  chlorine  to  form  sodium  hypochlorite,  (Na  O  Cl)  which 
becomes  active  in  the  sterilization  of  the  water." 

Pharmacists  find  considerable  demand  for  distilled  water  for  drinking 
purposes  as  well  as  for  use  in  dispensing.  However,  some  of  the  leading 
authorities  declare  that  drinking  distilled  water  is  objectionable,  because 
of  the  disturbance  of  the  osmotic  pressure  in  the  cells  of  the  digestive  tract. 
That  is,  the  distilled  water  acts  as  a  mechanical  poison.  There  is  an  ex- 
cessive endosmosis  inducing  an  abnormal  distention  of  the  cells,  causing 
physiological  disturbances.  This  action  is  due  to  the  fact  that  the  mineral 
salts  present  in  natural  drinking  water  are  absent  in  distilled  water. 

The  pharmacist  can  prepare  cheaply  and  simply  a  marketable  drinking 
water  which  does  not  have  the  objectionable  qualities  above  referred  to. 
Instead  of  distilling  the  water,  filter  it,  using  a  Pasteur-Chamberland  filter. 
Whether  a  large  or  small  filter  is  used  will  depend  upon  the  number  of 
customers  to  be  supplied.  In  all  probability  a  two-  or  three- tube  filter  is 
large  enough  for  the  average  retail  store.  "  Rapid  safety  filters  "  are  of  no 
value  whatever,  and  should  not  be  used,  as  they  are  in  no  sense  germ-proof. 
They  merely  remove  the  coarse  filth.  It  is  true  that  the  Pasteur- 
Chamberland  filters  are  not  absolutely  germ-proof,  but  they  remove  most 
of  the  microbes  present,  as  may  be  determined  bacteriologically  by  the 
pharmacist  himself.  The  few  germs  which  may  pass  through  the  filter 
are  killed  by  heating  the  water  to  the  boiling-point  or  30  minutes.  Such 
filtered  and  heat  sterilized  water  should  be  sold  in  large  sterile  glass  or 
earthenware  containers.  It  is  more  palatable  than  distilled  water  and 
does  not  interfere  with  the  osmotic  balance  of  cells. 

5.  Food  Preservatives 

The  use  of  food  preservatives  is  as  old  as  the  history  of  man.  Since 
remotest  antiquity  man  has  found  it  necessary  to  accumulate  a  supply  of 
food  during  the  seasonal  periods  of  plenty  in  order  to  tide  over  the  periods 
of  scarcity.  The  very  first  observation  made  was  that  the  accumulated 
and  stored  food  soon  showed  a  tendency  to  undergo  decomposition.  The 
next  observation  no  doubt  was  that  under  certain  conditions  some  organic 
food  kept  better  than  under  other  conditions,  thus,  for  example,  primitive 
man  gradually  learned  that  sun-dried  meats  did  not  decompose  nearly  as 
quickly  as  undried  meats.  No  doubt  the  value  of  smoking  meats  was  soon 
ascertained,  in  all  probability  purely  accidentally,  from  meats,  etc., 
which  had  been  exposed  to  the  smoke  of  the  camp  fire.  The  preservative 
value  of  heat,  as  in  cooking  and  roasting,  was  noted.  Next,  no  doubt  the 
preservative  properties  of  certain  chemicals  used  with  foods,  as  ashes 


35°  PHARMACEUTICAL  BACTERIOLOGY 

from  the  camp  fire,  salt,  brine,  vinegar,  wine  (alcoholic  beverages)  and 
sugar  was  noted.  Thus  primitive  man  made  use  of  the  germicidal  powers 
of  sunlight,  drying,  dry  heat,  moist  heat,  wood  ash,  smoke,  creosote  (in 
smoking  meats),  salt  solutions,  acids  (in  vinegar)  and  alcohol,  without 
having  any  idea  as  to  why  these  agents  retarded  or  prevented  the  decom- 
position of  organic  food  substances. 

In  modern  times  the  use  of  food  preservatives  is  based  upon  the  germ 
theory  of  decomposition.  The  time-honored  preservatives  above  referred 
to  have  continued  in  use  and  many  new  ones  have  been  added,  as  benzoic 
acid,  sodium  benzoate,  boracic  acid,  borax,  salicylic  acid,  sodium  sulphite, 
sulphurous  acid,  formalin  and  many  others.  A  somewhat  generalized 
theoretical  assumption  is  that  the  chemical  preservatives  in  foods  are  more 
or  less  injurious  to  health.  It  cannot  be  denied  that  some  of  the  preserva- 
tives used  are  irritating  to  the  kidneys  and  skin  and  some  perhaps  interfere 
more  or  less  with  food  digestion  and  assimilation.  It  has  long  been  known, 
for  example,  that  the  prolonged  consumption  of  salted  meats  produces 
serious  skin  affections  designated  as  scurvy.  The  sulphites  are  irritating 
to  the  kidneys;  formalin  interferes  with  digestion  of  foods,  etc.  However, 
there  can  be  little  doubt  that  in  the  comparative  sense  it  is  far  more  con- 
ducive to  health  and  longevity  to  eat  preserved  foods  than  foods  which  are 
more  or  less  decomposed.  We  are  daily  making  use  of  foods  which  contain 
small  quantities  of  natural  preservatives.  Cranberries,  for  example,  con- 
tain benzoic  acid;  formalin  and  phloroglucin  are  present  in  minute  quan- 
tities in  certain  plants;  a  multitudinous  variety  of  salts,  acids,  sugars, 
aromatic  oils,  etc.,  are  present  in  food  plants.  Food  chemists  do  not 
appear  to  be  seriously  worried  about  these  natural  preserving  agents  nor 
about  the  old-time  artificial  preservatives  as  smoke  creosote,  salt,  brine, 
sugar,  and  vinegar,  and  it  is  reasonable  to  suppose  that  careful  investiga- 
tion will  disclose  new  chemical  preservatives  which  are  superior  to  those 
mentioned.  The  whole  discussion  regarding  artificially  added  chemical 
food  preservatives  will  no  doubt  simmer  down  to  the  following:  What 
is  the  smallest  amount  of  the  least  objectionable  chemical  food  preservatives 
•which  must  be  added  to  certain  food  substances  in  order  to  preserve  them 
until  they  are  to  be  consumed?  Also  the  following  correlative  rule  should 
hold  good:  No  chemical  food  preservatives  whatsoever  should  be  used  as 
such  excepting  in  cases  where  modern  methods  of  heat  and  cold  sterilization 
and  preservation  fail  or  are  inapplicable. 

The  use  of  sugar  and  of  salt  in  moderation  are,  of  course,  always  permis- 
sible, since  these  substances  are  essentials  in  many  foods.  The  objection 
and  danger  in  the  use  of  food  preservatives  lie  in  the  fact  that  careless 
manufacturers  are  too  prone  to  use  them  in  order  to  avoid  employing 
harmless,  though  perhaps  less  simple,  and  more  expensive  means  of  food 


DISINFECTANTS   AND   DISINFECTION  351 

preservation.  Chemical  preservatives  make  it  possible  for  the  unscrupu- 
lous to  use  decomposed  and  otherwise  objectionable  food  material.  Fur- 
thermore, there  is  a  strong  tendency  to  use  chemical  preservatives  in 
excess,  in  spite  of  the  strictest  legal  quantitative  limitations. 

The  following  is  a  brief  summary  of  the  more  common  food  preserva- 
tives and  their  use. 

The  physical  and  mechanical  means  of  food  preservation  have  been 
referred  to,  likewise  the  use  of  heat,  cold,  smoke,  etc.  One  of  the  most 
satisfactory  methods  of  preserving  foods,  now  employed  in  all  up  to  date 
canneries,  is  a  combination  of  heat  sterilization  with  air  exclusion  (air 
pump  and  by  displacement).  The  food  products  as  meat,  corn,  beans, 
asparagus,  peas,  jams,  jellies,  preserves,  etc.,  etc.,  are  heated  (100°  C.  to 
120°  C.)  to  destroy  all  germ  life,  the  containers  (tins,  glass)  are  also  heated 
and  then  nearly  filled  to  exclude  as  much  air  (oxygen)  as  possible.  Air 
(oxygen)  is  necessary  for  the  growth  of  bacteria,  yeasts  and  molds,  hence 
a  well  filled  container,  with  a  minimum  of  oxygen  is  less  likely  to  show 
decomposition  effects  ("swells,"  " leaks")  than  containers  which  are  not 
well  filled.  It  is  claimed  that  wholesome  fruit,  meat,  etc.,  (free  from 
decomposition),  which  is  well  sterilized  by  steam  heat  and  put  up  in  well 
sterilized  containers  requires  no  chemical  preservative  whatever.  It  is, 
however,  customary,  in  the  case  of  fruits,  to  add  sugar  as  a  preservative 
and  also  for  the  purpose  of  rendering  the  article  more  palatable.  The 
sugar  from  sugar  cane  is  quite  universally  used  in  preference  to  the  sugar 
from  the  sugar  beet.  This  is  no  doubt  due  to  the  fact  that  sugar  beet 
sugar  contains  slightly  more  organic  impurities  and  is,  hence,  under 
similar  methods  of  use  as  to  quantity,  degree  of  heat  sterilization,  etc., 
slightly  more  likely  to  undergo  decomposition. 

Preservation  of  food  substances  by  drying  is  coming  into  use  more  and 
more.  By  this  method  it  is  possible  to  keep,  for  variable  periods  of  time,  a 
great  variety  of  foods  as  apples,  peaches,  pears,  bananas,  potatoes  and 
many  other  vegetables,  besides  bread,  meats,  eggs,  milk  and  other  sub- 
stances, which  were  formerly  more  generally  preserved  by  the  canning 
method.  Eggs  may  also  be  preserved  entire  by  giving  them  a  coating  of 
tallow,  wax,  paraffin  or  soluble  silicate,  which  exclude  the  air,  or  they  may 
be  preserved  in  brine,  salt  or  other  so-called  harmless  chemical  preservative. 

Herring,  cod  and  other  fish  are  often  preserved  in  a  brine  of  salt  or  of 
equal  parts  of  salt  and  borax  or  boric  acid.  Of  meats,  fish  is  particularly 
liable  to  decomposition  and  it  is  declared  that  certain  kinds  cannot  be 
preserved  in  salt  alone,  that  it  is  necessary  to  add  boric  acid,  rubbing  the 
preservative  well  into  incisions  made  along  the  spinal  column  where  the 
decomposition  develops  earliest.  Salt  is  used  with  meats  generally  and  with 
butter.  Two  per  cent,  of  salt  in  butter  is  sufficient,  though  as  much  as 


352  PHARMACEUTICAL  BACTERIOLOGY 

15  per  cent,  and  more  is  sometimes  added  to  increase  the  weight.  A  com- 
bination of  salt  and  saltpeter  is  added  to  meat  (brine).  The  saltpeter 
gives  a  red  tint  to  meat  besides  serving  as  a  preservative.  Saltpeter  is 
considered  more  or  less  injurious  to  health,  when  taken  with  food  to  the 
amount  of  0.5  of  i  per  cent,  or  more. 

Borax  and  boric  acid  is  often  added  to  milk.  4.4  grains  to  the  pint 
(0.05  per  cent.)  keeps  milk  sweet  for  a  time  (10  to  14  hours  and  longer). 
Small  doses  of  borax  and  boric  acid  (up  to  i  gram  per  day)  is  considered 
harmless.  Certain  preservatives  of  a  proprietary  nature  as  "Preserving 
Salts,"  "Preservative,"  consist  of  borax  and  salt  in  the  proportion  of  three 
to  one. 

Formalin  (the  40  per  cent,  commercial  solution)  added  to  milk,  to  the 
amount  of  1-50,000,  retards  souring  for  several  hours;  1-1.0,000  prevents 
souring  for  twelve  hours  and  longer,  and  in  this  amount  it  does  perhaps 
very  little  harm,  though  it  is  believed,  due  to  its  coagulating  effects,  to  in- 
terfere with  the  digestibility  of  milk;  particularly  in  children.  Several 
marketed  milk  preservatives  have  formalin  for  their  principal  ingredient 
("milk-sweet,"  "iceline,"  "freezine"). 

Sulphurous  acid  and  sulphites  are  added  to  vinegar,  pickles,  catsups, 
etc.,  anchovy  pastes,  canned  and  dried  fruits,  etc.,  to  the  amounts  of  0.2 
to  1.15  per  cent.  The  part  active  as  a  preservative  is  the  available  SOz 
which  is  gradually  oxidized  into  sulphates.  These  agents  are  deodorant, 
as  well  as  preservative,  because  of  the  high  oxidizing  power. 

Butchers  use  sulphite  preservatives  to  dust  over  sausage  meats  for  the 
double  purpose  of  giving  the  meat  a  red  color  (due  to  the  O  combining 
with  the  hemaglobin  of  the  blood)  and  to  destroy  possible  odors  of  decom- 
position. 0.05  per  cent,  of  sulphites  is  sufficient  to  check  decomposition  in 
fresh  meats,  though  the  best  results  follow  the  use  of  0.5  per  cent.  0.2  per 
cent,  has  germicidal  powers  when  combined  with  cold.  Sometimes 
aniline  color  is  added  to  the  sausage  meat  preservatives. 

Sodium  benzoate  is  perhaps  the  most  extensively  employed  preserva- 
tive and  at  the  same  time  the  least  harmful,  o.i  per  cent,  added  to  food 
articles,  as  meats,  fruits,  catsups,  vinegar,  cider,  etc.,  checks  decomposition. 
Generally,  however,  more  than  o.i  per  cent,  is  added,  from  0.2  to  0.5  per 
cent.  The  percentage  of  benzoate  preservative  is  likely  to  vary  because 
of  its  volatile  nature;  canners  quite  generally  add  an  excess  knowing  that 
much  of  it  will  be  carried  off  with  the  vapors  escaping  during  the  heating 
process.  As  a  result  it  follows  that  products  declared  to  contain  o.i  per 
cent,  of  benzoate  may  upon  chemical  examination  show  the  actual  amounts 
to  range  from  a  mere  trace  (0.05  per  cent,  to  0.5  per  cent,). 

Next  to  benzoate,  salicylic  acid  is  perhaps  the  most  common  food  pre- 
servative, used  much  like  benzoate,  in  strengths  varying  from  .01  to  0.25 


DISINFECTANTS   AND   DISINFECTION  353 

per  cent.  It  is  frequently  added  to  beers,  cordials,  wines  and  foods  (2  to 
4  grains  to  the  pint)  containing  sugars.  It  is  also  used  as  a  surgical 
dressing,  but  other  less  irritating  wound  disinfectants  are  given  the 
preference. 

Crude  pyroligneous  acid  is  used  as  a  meat  preservative.  This  acid  is 
obtained  by  the  destructive  distillation  of  wood  and  contains  creosote  and 
other  tarry  matter  and  imparts  the  odor  and  taste  of  smoked  products. 
Meats,  fish,  etc.,  are  immersed  in  a  solution  of  this  acid,  dried  and  sold  as 
smoked.  This  constitutes  the  "quick"  or  "dip"  method  of  smoking 
meats  as  compared  with  the  usual  slower  method  of  exposing  the  meats  to 
the  smoke  of  slowly  burning  wood. 

The  following  are  a  few  of  the  less  commonly  employed  preservatives : 
Fluorine  compounds  are  used  in  strengths  of  from  0.03  to  0.02  per  cent. 
Alum  is  sometimes  used  in  pickling  vegetables  and  meats  (brine)  because 
of  the  hardening  effects  produced.  Copper  sulphate  is  much  used  in 
pickling  cucumbers,  peas,  string  beans  and  other  green  vegetables  for  the 
purpose  of  deepening  the  green  color.  Sodium  and  calcium  carbonate  are 
sometimes  added  to  cider  and  wine  to  check  the  souring  process  (by  com- 
bining with  the  fruit  acids).  Formic  acid  is  a  powerful  preservative. 
0.014  to  0.08  per  cent,  retards  fermentation.  Saccharin,  sucrol  and  dulcin 
are  sweetening  as  well  as  preserving  agents.  Peroxide  of  hydrogen  is  used 
as  a  preservative.  It  is  also  a  deodorant.  The  use  of  saccharin  in  food  is 
no  longer  permissible  in  the  United  States. 

6.  Insecticides  and  Other  Pest  Exterminators 

The  farmer,  fruitgrower  and  florist  have  many  enemies  belonging  to 
the  insecta  and  to  other  divisions  of  the  animal  kingdom,  which  interfere 
with  the  productiveness  of  crops.  The  remedies  employed  against  these 
pests  are  numerous.  We  shall  mention  only  a  few  of  the  more  useful  ones, 
explaining  their  action  very  briefly.  They  may  be  grouped  into  powders, 
gases,  sprays  and  washes. 

A.  Powders. — These  may  be  applied  by  the  "pepper  box"  method,  the 
material  being  placed  in  a  box,  usually  of  tin,  with  perforations,  through 
which  the  powder  sifts  on  shaking.  Or  a  blowing  device  may  be  used, 
like  the  ordinary  bellows  box  for  blowing  insect  powder,  or  modifications 
of  this  simple  device.  A  third  method  known  as  the  sifting  method  is 
much  in  vogue  in  the  cotton  fields.  The  powder  is  placed  in  a  porous  bag 
or  cloth,  fastened  to  a  stick  and  shaken  over  the  plants  to  be  treated. 
Only  three  powders  are  used  to  any  considerable  extent,  as  follows: 

a.  Slaked  Lime. — Dry  air  slaked  lime  is  reduced  to  a  uniformly  fine 
powder  which  is  then  ready  for  use.  It  is  very  efficacious  with  all  slimy 
animals,  as  slugs  and  snails.  It  is  applied  to  plants  when  the  pests  are 
23 


354  PHARMACEUTICAL  BACTERIOLOGY 

active,  that  is,  in  the  early  morning  or  in  the  evening.     Lime  is  used  where 
paris  green  is  not  permissible,  as  with  fruit  plants  and  edible  herbs. 

b.  Sulphur. — The  flower  of  sulphur  or  ground  sulphur  is  a  very  widely 
used  remedy  for  fungous  pests,  as  mildew;  also  for  the  red  spider  and 
thrips.     Sulphur  is  active  only  in  the  sunlight,  particularly  on  a  hot  day. 

The  flower  of  sulphur  gives  better  results  than  the  ground  sulphur  be- 
cause it  "sticks"  better.  It  should  be  applied  evenly  and  not  too  thickly. 
Remember  that  sulphur  dioxide  is  very  injurious  to  plants,  therefore 
fumigation  by  burning  sulphur  is  out  of  the  question. 

c.  Paris  Green  and  Other  Arsenicals. — These  are  generally  not  used  in 
the  dry  powdered  form.     When  so  used  they  are  diluted  with  flour,  dust 
or  other  inert  powdered  material.     Must  be  sparingly  applied  and  evenly 
distributed,  otherwise  serious  damage  may  be  done  to  the  foliage. 

B.  Gases. — Gases  diffuse  with  great  rapidity  and  when  applied  within 
an  enclosed  space  will,  in  a  short  time,  be  uniformly  distributed  throughout 
the  enclosed  space.  The  rapid  diffusion  of  gases  is  a  great  hindrance  to 
their  practical  utilization  in  the  open  as  in  orchards,  fields  and  gardens. 
Their  use  is  quite  limited. 

a.  Carbon  Bisulphide. — This  is  not  used  with  growing  plants  though  it 
is  applied  to  stored  seeds,  and  dry  plants  and  grains,  for  the  purpose  of 
killing  insects  and  other  destructive  animals.  It  is  also  used  to  kill  pests 
which  live  in  the  soil,  as  the  grape  Phylloxera.  For  this  purpose  a  machine 
is  used  which  injects  the  bisulphide  into  the  soil.  To  destroy  pests  in  drug 
plants,  seeds  and  grain,  enclose  them  in  a  space,  place  a  dish  containing 
the  bisulphide  on  top  of  the  material.  The  vapor  being  heavier  than  the 
air,  gravitates  downward  and  soon  fills  the  entire  enclosed  area.  The 
amount  necessary  to  do  the  work  will  depend  upon  the  nature  of  the  ma- 
terial to  be  treated  and  the  tightness  of  the  enclosure.  Roughly  estimated 
a  dram  of  the  carbon  bisulphide  to  five  pounds  of  the  material  is  sufficient. 
Grainmen  usually  apply  one  pound  to  the  ton  of  grain,  if  the  bin  is  tight. 

Carbon  bisulphide  is  one  of  the  most  effective  remedies  against  the 
gopher  and  the  ground  squirrel.  Use  the  remedy  after  a  rain  as  the  soil 
is  then  less  porous.  Pour  an  ounce  over  a  rag  or  other  porous  substance 
(horse  droppings  are  much  used),  stuff  this  into  the  hole  and  plug  with  a 
ball  of  dirt.  The  bisulphide  is  also  used  to  kill  the  yellow-jacket,  which  is 
very  injurious  to  fruit,  also  the  root  crown  borer  of  the  peach,  and  to  disin- 
fect grapevine  cuttings,  etc.,  etc. 

b.  Hydrocyanic  Acid  Gas. — This  is  about  the  only  gas  which  is  powerful 
enough  to  kill  insects  and  yet  not  injure  the  foliage.  It  is  used  by  covering 
the  tree,  shrub  or  bush  with  a  tent  cloth  or  canvas  which  should  be  oiled  to 
keep  in  the  vapor.  The  vessel  containing  the  chemicals  is  placed  under- 
neath. Exposure  of  from  thirty  to  fifty  minutes  is  usually  sufficient. 


DISINFECTANTS   AND   DISINFECTION  355 

About  one  ounce  of  potassium  cyanide  to  150  cubic  feet  of  space  is  required. 

The  gas  is  extremely  poisonous  and  is  often  destructive  to  foliage.  It  is 
preferably  applied  at  night  as  it  is  then  less  injurious  to  the  foliage. 

C.  Sprays  and  Washes. — Plant  pests  are  most  generally  destroyed  by 
spraying  agents  or  washes.  A  wash  is  really  a  more  liberal  application  of 
the  spray,  the  two  being  alike  as  to  the  results  to  be  attained  from  their  use. 

For  low  plants  the  remedy  can  be  applied  by  means  of  a  sprinkling  can 
but  the  better  method  is  to  use  some  form  of  force  pump  with  spray  nozzle. 
A  good  spray  pump  should  maintain  a  uniformly  constant  as  well  as  ade- 
quate pressure,  should  be  simple  of  construction,  with  all  parts  readily 
replaceable.  The  nozzle  should  break  up  the  stream  into  a  fine  mist. 

It  is,  of  course,  desirable  to  get  as  much  as  possible  of  the  spray  to 
remain  on  leaf  or  stem  and  to  have  it  evenly  distributed.  If  put  on  too 
abundantly  the  fine  droplets  or  gobules  on  the  leaf  will  run  together  and 
roll  off  to  the  ground.  The  nozzle  must  not  be  held  near  the  plant  to  be 
sprayed  in  order  to  get  the  desirable  dew-like  deposit  on  the  leaf. 

For  scale  insects  a  thorough  moistening  is  necessary,  wetting  the  bark, 
the  scale  and  eggs.  In  order  to  accomplish  this  the  nozzle  must  be  held 
close 

The  following  table  by  Woodworth  will  indicate  the  method  of  pre- 
paring and  using  the  more  important  spraying  solutions: 

The  well  known  Bordeaux  mixture,  so  extensively  used  as  a  spray  and 
wash  is  prepared  as  follows: 

Water,  50  gal. 

Copper  sulphate,  6  Ib. 

Unslaked  lime,  4  Ib. 

The  adhesive  properties  can  be  increased  by  adding  soft  soap  in  quantity 
equal  to  that  of  the  copper  sulphate.  It  is  also  advisable  to  dilute  the 
mixture  for  spring  spraying.  It  is  the  most  effective  and  perhaps  the 
cheapest  fungicide  that  can  be  used. 

Aphides  (plant  lice)  and  similar  plant  parasites  may  also  be  destroyed 
with  weak  solutions  of  alum  (1.5  to  2  per  cent.).  Beetles  may  be  killed  by 
sprinkling  a  mixture  of  equal  parts  of  red  lead,  sugar  and  flour,  near  their 
hiding  places,  or  a  mixture  of  borax  20  parts  and  precipitated  carbonate 
of  baryta  (native  witherite  will  not  answer  the  purpose).  A  great  variety 
of  substances  are  recommended  for  the  extermination  of  ants,  as  borax, 
camphor,  balsam  of  Peru,  spraying  with  benzine,  etc.  In  lawns  and  in  the 
open  generally  (in  ant  hills)  they  are  most  quickly  destroyed  by  means  of 
carbon  bisulphide.  This  kills  the  ants  as  well  as  the  larvae. 

The  exterminators  for  pests  of  all  sorts  is  legion  and  those  especially 
interested  must  consult  some  standard  work  on  formulas  such,  as  the  Scien- 
tific American  Cylcopedia  of  Formulas  (Hopkins). 


356 


PHARMACEUTICAL  BACTERIOLOGY 


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CHAPTER  XVI 
STERILIZATION   AND  DISINFECTION    IN  THE  PHARMACY 

It  is  only  within  very  recent  years  that  sterilization  in  the  pharmacy 
has  received  any  serious  attention.  Certain  pharmacopoeias,  notably  those 
of  Austria  and  Belgium,  give  specific  directions  regarding  the  sterilization 
of  certain  medicamenta,  particularly  those  intended  for  hypodermic  use. 
The  German,  English,  Italian,  Swiss  and  other  pharmacopoeias  give  direc- 
tions regarding  certain  sterilizing  processes  which  may  be  applied  to  a  few 
articles.  Fischer,  Stich,  Deniges,  Mario,  Schoofs  and  other  European 
investigators  have  given  the  subject  much  attention  and  have  perfected 
many  of  the  details  of  procedure. 

Some  of  the  non-official  methods  of  sterilization  are  of  very  doubtful 
practicability.  Particularly  the  methods  recommended  for  the  steriliza- 
tion of  pharmaceutical  solutions  by  means  of  the  ultra-violet  rays  and  by 
means  of  chemical  disinfectants.  Lesure  sums  up  the  use  of  the  ultra- 
violet rays  as  follows:  "A  series  of  experiments  shows  that,  at  present,  the 
ultra-violet  rays  can  scarcely  be  regarded  as  a  practical  means  of  sterilizing 
pharmaceutical  solutions,  such  as  hypodermic  injections.  It  is  not  yet 
possible  to  sterilize  liquids  in  small  closed  glass  vessels,  since  the  glass 
absorbs  the  rays  of  shortest  wave  length,  which  are  precisely  those  of  most 
active  sterilizing  power.  Possibly  on  a  large  scale  solutions  could  be 
sterilized  in  bulk  and  then  filled,  in  vacua,  into  sterilized  small  receivers. 
The  rays  might  be  useful  for  substances  which  are  decomposed  by  treat- 
ment in  the  autoclave.  Some  substances  are,  however,  so  readily  de- 
composed by  ultra-violet"  rays,  that  their  solutions  can  never  be  sterilized 
therewith.  Such  are  solutions  of  quinine  salts,  of  mercuric  iodide,  of 
atoxyl,  of  eserine,  of  apomorphine  and  some  glucosides,  as  for  example 
gentiopicrin.  Opaque  solutions  and  suspensions  of  solids  cannot  be  thus 
sterilized.  The  permeability  of  the  different  solutions  to  the  rays  also 
varies  very  greatly.  Apart  from  the  question  of  decomposition,  it  is  found 
that,  in  the  case  of  gentiopicrin,  completely  sterile  solutions  were  not  ob- 
tained even  after  an  exposure  of  half  an  hour;  on  the  other  hand  ancubin 
solutions  were  completely  sterilized  in  thirty  seconds."  The  decomposi- 
tion changes  due  to  the  ultra-violet  rays  are  not  clearly  understood.  The 
indications  are  that  there  are  no  very  marked  chemical  changes  in  such 
substances  as  cocaine  and  pilocarpin  hydrochloride  after  three  hours' 
exposure.  Arbutin  shows  a  change  in  a  few  minutes.  There  is  so  much 

357 


358  PHARMACEUTICAL  BACTERIOLOGY 

uncertainty  as  to  the  results  that  the  method  cannot  as  yet  be  recom- 
mended for  practical  use. 

The  addition  of  disinfectants  to  medicines  for  purposes  of  sterilization 
has  recently  received  some  attention.  The  use  of  formaldehyde,  ether, 
chloroform  and  alcohol,  have  been  recommended,  each  having  its  special 
use  in  practice.  The  general  criticisms  made  regarding  the  use  of  the  ultra- 
violet rays  also  apply  here.  Currie  recommends  a  formalin  method  as  fol- 
lows: applicable  to  infusions  of  calumba,  gentian,  quassia  and  senega. 
"The  infusions  of  calumba  and  quassia  are  simply  evaporated  to  one- 
eighth  of  their  bulk,  filtered,  and  4  minims  of  the  ordinary  40  per  cent,  solu- 
tion of  formaldehyde  added  to  each  fluid  ounce  of  the  concentrated  infusion. 
On  dispensing,  the  requisite  amount  is  put  in  a  shallow  basin  and  brought 
sharply  to  the  boil,  thus  dissipating  the  formaldehyde.  The  infusion  is 
then  diluted  to  the  normal  strength  with  sterilized  distilled  water. 
Infusion  of  gentian  is  made  from  gentian  root  alone,  and  concentrated. 
To  this  is  added  essence  of  lemon  (i  in  10),  and  the  official  tincture  of 
orange  in  the  proportion  of  2  fluid  drams  of  the  former  and  i  fluid  ounce  of 
the  latter  to  each  pint  of  the  infusion.  There  is  also  added  4  minims  of 
40  per  cent,  solution  of  formaldehyde  to  each  fluid  ounce  of  infusion. 
Infusion  of  senega  is  concentrated  by  evaporation  and  to  prevent  precipita- 
tion, 5  grains  of  potassium  bicarbonate  are  added  to  each  fluid  ounce  of  the 
concentrated  solution,  and  4  minims  of  40  per  cent,  solution  of  formal- 
dehyde. In  case  of  both  gentian  and  senega  infusion,  the  formaldehyde 
is  dissipated  at  the  time  of  dispensing,  in  the  manner  already  described. 
The  advantages  of  this  process  are  ease  of  manipulation,  cheapness,  and 
the  certainty  of  the  antiseptic  condition  of  the  infusion  while  being  kept  in 
stock  and  until  dispensed.  The  quantity  of  formaldehyde  remaining  in 
the  diluted  infusion  is  infinitesimal,  and  may  be  ignored  for  all  practical 
purposes." 

It  is  known  that  weak  solutions  of  hypodermic  and  intravenous  solu- 
tions, unless  sterilized,  will  show  numerous  bacteria  upon  standing  for  a 
time.  One  per  cent,  solutions  of  pilocarpin,  atropin,  cocaine,  morphine, 
and  fluid-extract  of  ergot  have  been  found  to  contain  millions  of  bacteria 
per  cc.  However,  10  per  cent,  iodoform  glycerin,  camphorated  oil  (i  in  10, 
solutions  of  apomorphin  (0.2  in  20),  quinine  (i  in  10),  antipyrin  (5  in  10), 
cocaine  (10  per  cent.)  are  usually  quite  free  from  bacteria.  In  a  general 
way  the  bacterial  content  of  medicinal  solutions  decreases  directly  with  the 
degree  of  concentration.  Pus  microbes  die  at  once  in  ether  and  in  a 
saturated  solution  of  quinine,  whereas  they  remain  active  in  a  10  per  cent, 
solution  of  cocaine.  A  2  per  cent,  solution  of  morphine  kills  pus  microbes 
in  twenty-four  hours,  while  pure  glycerin  kills  them  only  after  an  exposure 
of  six  to  eight  days. 


STERILIZATION  AND   DISINFECTION  IN  THE  PHARMACY       359 

A  perfectly  safe  rule  for  the  pharmacist  is  to  consider  all  medicamenta 
which  he  handles  and  which  he  may  be  called  upon  to  dispense,  as  being 
possibly  contaminated  and  to  sterilize  and  disinfect  all  articles  which  in  his 
judgment  as  a  qualified  pharmacist  may  require  such  treatment,  in  so  far 
as  it  is  practically  possible.  The  retail  pharmacist  must  not  place  too 
much  confidence  in  the  assertions  of  comparatively  little  known  manufac- 
turers and  wholesale  houses,  regarding  the  sterile  conditions  of  the  articles 
which  they  may  supply. 

The  medicines  found  in  a  drug  store  and  dispensed  by  the  pharmacist 
may  be  grouped  as  follows : 

A.  Medicines  which  do  not  Generally  Require  Sterilization 

a.  For  internal  administration  per  mouth.    They  may  be  contami- 
nated or  may  become  contaminated  on  standing  for  a  time.     Such  medi- 
cines should  be  rejected.     Do  not  attempt  to  render  them  usable  by 
sterilization. 

b.  Mouth  washes  and  gargles. 

c.  Enemas.     Enemas  for  young  children  and  such  enemas  as  are  to  be 
applied  to  inflamed  or  otherwise  pathologic  conditions  of  the  intestinal 
mucous  membrane,  should  be  sterilized. 

d.  Medicamenta  which  are  to  be  applied  to  the  intact  skin,  or  to  the 
scalp. 

B.  Medicines  Which  Require   Sterilization 

a.  Those  intended  for  intravenous  and  hypodermic  use.     Not  only 
must  these  be  absolutely  sterile  but  they  must  be  in  perfect  solution,  before 
using. 

b.  Those  to  be  applied  to  cuts,  bruises,  abrasions,  wounds,  ulcers, 
sores,  and  to  the  broken  skin  generally. 

c.  Those  to  be  applied  to  inflamed  mucous  membranes,  as  enemas, 
douches,  etc. 

d.  Solutions  for  the  irrigation  of  the  bladder. 

e.  Eye  medicines,  as  washes  and  other  solutions,  intended  for  direct 
application  to  the  eye. 

i.  Methods  of  Sterilization 

The  following  methods  of  sterilization  are  applicable  in  the  pharmacy 
and  should  be  consistently  practised: 

A.  Sterilization  of  Containers. — The  glassware  and  other  containers 
used  in  the  pharmacy  should  be  cleaned  and  sterilized  as  follows: 

a.  Bottles  and  Glassware  Generally. — Wash  and  rinse  in  warm  water  to 
remove  dust,  dirt,  sand,  straw,  etc.,  then  wash  and  rinse  in  hot  water  with 
2  to  5  per  cent,  sodic  hydrate.  Neutralize  the  sodic  hydrate  by  washing 


360  PHARMACEUTICAL  BACTERIOLOGY 

and  rinsing  in  2  to  5  per  cent,  hydrochloric  acid.  Finally  wash  and  rinse 
in  hot  sterile  water  and  allow  to  drain.  Wipe  dry  and  plug  lightly  with 
cotton.  Place  the  plugged  bottles  in  a  hot-air  sterilizer  and  heat  for  one 
hour  at  130°  C.  to  140°  C.  Keep  these  cleaned,  sterilized,  and  cotton 
plugged  bottles  in  clean  container  in  a  dry  clean  store-room,  until  wanted 
for  use. 

b.  Porcelain  and  Similar  Containers. — May  be  cleaned  and  sterilized 
like  glassware.     Plugging  with  cotton  is  as  a  rule  inadmissible. 

c.  Large  Flasks,  Jugs,  Etc. — Large  containers  are  as  a  rule  difficult 
to  sterilize  and  for  this  very  reason  are  often  subject  to  special  neglect. 
Proceed  much  as  for  bottles,  observing  greater  caution  as  to  changes  in 
temperature.    Large  bottles,  carboys  and  similar  containers  cannot  be 
sterilized  by  means  of  boiling  hot  water  as  they  are  very  apt  to  crack. 
They  may  be  sterilized  by  means  of  carbolic  acid  (5  per  cent.),  lysol  (1.5 
per  cent.)  or  formaldehyde  (4  per  cent.),  then  thoroughly  rinsed  in  sterile 
water,  allowed  to  drain,  plugged  with  cotton,  carefully  heated  in  hot-air 
sterilizer  for  one  hour  or  more  at  115°  to  120°  C.     Cool  gradually. 

d.  Tin  Containers. — Wash  and  rinse  thoroughly  in  water;  boil  for 
thirty  minutes,  drain  and  dry  and  sterilize  in  dry-air  sterilizer  for  one 
hour  at  100°  C. 

B.  Sterilization  of  Apparatus  and  Tools. — It  is  of  the  highest  impor- 
tance that  mortar  and  pestle,  spatulas,  percolators,  pill  and  suppository 
machines,  mixing  plates,  etc.,  etc.,  should  be  clean  and  sterile.     This 
means  a  liberal  use  of  hot  water,  green  or  soft  soap,  and  clean  towels. 
The  sink,  the  floor  of  the  dispensing  room,  the  tables,  chairs,  desks,  in  fact 
everything  in  and  about  the  dispensing  room  should  be  scrupulously  clean. 

C.  Sterilization  of  Corks  and  Other  Stoppers  for  Containers. — It  would 
be  energy  wasted  to  clean  and  sterilize  the  containers  if  the  stoppers  are  not 
also  clean  and  sterile.     Sterilize  corks  by  washing  in  hot  60  to  75  per  cent, 
alcohol,  drain  and  heat  in  hot-air  sterilizer  for  one  hour  at  130°  C.     Keep 
these  corks  in  sterilized  wide-mouthed  ground-glass  capped  bottles.     Take 
out  corks  as  wanted  by  means  of  a  sterile  pair  of  pincers,  not  by  means  of 
fingers.     Other  stoppers,  as  of  glass,  of  wood,  of  rubber,  must  also  be 
cleaned  and  sterilized.     Rubber  caps,  rubber  stoppers,  and  other  rubber 
goods  may  be  sterilized  by  boiling  in  water  for  thirty  minutes. 

D.  Sterilization  of  Surgical  Supplies. — a.  Bandaging  materials,  cotton, 
absorbent  gauze,  etc.,  may  be  sterilized  by  wrapping  in  cheese  cloth  or 
filter  paper,  first  placing  a  grain  of  f uchsin  or  other  aniline  dye  in  the  center 
of  the  package  (wrapped  in  paper  or  cloth),  and  sterilizing  in  steam  for  one 
hour.     The  dye  particle  is  introduced  as  a  test  object  to  ascertain  if  the 
steam  has  penetrated  the  entire  package.     If  it  has  penetrated  the  entire 
package  it  will  be  indicated  by  a  spreading  of  the  color.     Afterward,  dry 


STERILIZATION   AND    DISINFECTION    IN    THE    PHARMACY  361 

for  one  hour  at  100°  C.  in  the  hot-air  sterilizer.     For  this  purpose  the  form 
of  Arnold  steam  sterilizer  shown  in  Fig.  18  will  be  found  very  useful. 

b.  Sewing  materials,  such  as  needles,  forceps,  catgut,  etc.,  require 
careful  sterilization  before  using.     All  metal  instruments  and  appliances, 
including  silver  wire,  can  be  sterilized  in  5  per  cent,  carbolic  acid  if  nec- 
essary or  they  may  be  boiled  for  30  to  50  minutes.     Wipe  perfectly  dry 
with  sterile  towels  and  place  in  hot-air  sterilizer  for  one  hour  at  100°  C. 
In  order  to  keep  them  in  sterile  condition  for  immediate  use  they  must  be 
kept  wrapped  in  sterilized  cloth  or  cotton. 

c.  Catgut  requires  thorough  sterilization  as  not  infrequently  spores  of 
disease  germs  (as  anthrax)  are  present.     The  so-called  cumol  (cumene) 
method  of  catgut  sterilization  is  quite  generally  adopted  in  the  hospitals  of 
Germany  and  of  other  European  countries.     Wind  the  catgut  in  the  usual 
ring  form,  dry  in  hot-air  sterilizer  for  two  hours  at  70°  C.,  place  rings  in  a 
vessel  (beaker,  etc.)  with  cumol  on  sand-bath  and  heat  to  155°  C.  or  165°  C. 
(the  boiling-point  of  cumol),  turn  off  the  gas  and  allow  to  remain  in  the  hot 
cumol  for  one  hour.     The  cumol  dish  should  be  covered  with  a  fine  mesh 
wire  screen  to  guard  against  catching  fire.    Take  the  catgut  rings  out  of  the 
cumol  by  means  of  sterile  pincers  and  place  in  benzine.for  three  hours,  then 
allow  the  benzine  to  evaporate  in  sterile  Petri  dishes. 

d.  Silver  catgut  is  preferably  sterilized  in  i  per  cent,  silver  citrate 
(itrol)  or  i  per  cent,  silver  lactate  (actol),  allowing  it  to  remain  for  six  hours 
which  destroys  even  the  anthrax  spores.     Next  expose  the  catgut  to  light 
(in  sterile  dishes)  for  a  day  or  two,  then  wind  or  fasten  on  glass  and  pre- 
serve in  95  per  cent,  alcohol  with  10  per  cent,  glycerin.     Actol  and  itrol 
ionize  silver  far  less  actively  than  silver  nitrate,  hence  their  preference. 

e.  Catheters,  drainage  tubing  and  other  rubber  materials  are  sterilized 
by  boiling  in  water  with  5  per  cent,  sodic  hydrate.     Rubber  goods  will  not 
stand  prolonged  and  frequent  boiling.     Do  not  sterilize  metal  ware  with 
rubber  goods. 

e.  Sterilization  of  Medicines. — As  a  rule,  medicines  which  are  prepared 
under  aseptic  surroundings  and  conditions  do  not  require  sterilization. 
However,  the  ideal  conditions  rarely  exist  and  subsequent  sterilizations 
become  desirable  and  even  necessary. 

Tooth  powders,  dusting  powders  and  similar  substances  may  be  ster- 
ilized at  a  dry  temperature  of  70°  C.,  for  three  to  four  hours.  Salves  and 
pastes  are  difficult  to  sterilize.  Low  temperatures  (from  60°  C.  to  70°  C.) 
for  several  hours  may  be  employed. 

Solutions  for  subcutaneous  injection,  for  wound  irrigation,  for  bladder 
irrigation,  solutions  of  boric  acid,  of  tannic  acid,  aquae,  normal  salt  solu- 
tions and  all  weaker  solutions  of  chemicals,  intended  for  washes  and  irriga- 
tion in  surgery,  should  be  sterilized  by  boiling  for  five  minutes.  Strong 


362  PHARMACEUTICAL  BACTERIOLOGY 

solutions  of  chemicals  (as  acids,  alkalies,  etc.)  do  not  require  sterilization 
as  they  are  themselves  strongly  germicidal. 

Alkaloidal  and  glucosidal  solutions,  and  solutions  of  alkaloidal  salts, 
tinctures  and  fluidextracts,  should  be  carefully  filtered  and  sterilized  in 
sealed  containers  at  a  temperature  of  60°  C.,  one  hour  each  day  for  six 
days.  Concentrated  alkaloidal  solutions  may  be  similarly  sterilized.  It 
is  not  advised  to  employ  a  higher  temperature  for  these  substances  inas- 
much as  the  decomposition  changes,  if  any,  which  may  take  place  at  100°  C. 
are  not  clearly  understood.  To  be  on  the  safe  side,  the  lower  temperature 
(60°  C.)  should  be  employed. 

In  the  case  of  solutions  or  emulsions  for  hypodermic  use,  prepared  with 
oil,  the  oil  is  first  to  be  treated  with  alcohol  (95  per  cent.)  to  remove  the 
oleic  acid.  Oily  solutions  of  calomel,  yellow  oxide  of  mercury,  lecithin, 
and  of  camphor  are  to  be  prepared  with  sterile  materials,  then  placed  in  a 
boiling  water-bath  for  ten  minutes  or  in  an  air-bath  at  100°  C.  An  interest- 
ing requirement  is  exacted  by  the  Italian  Pharmacopeia  as  regards  the 
glass  of  the  containers  for  hypodermic  injections :  Ten  to  twelve  ampuls  or 
five  or  six  bottles  are  filled  with  a  clear  solution  of  i  per  cent,  mercuric 
chloride,  then  sealed.  They  are  then  left  in  an  autoclave  at  112°  C.  for 
half  an  hour,  at  the  expiration  of  which  time  no  brownish  turbidity  should 
be  perceptible. 

Some  of  the  points  pertaining  to  the  sterilization  of  alkaloidal,  gluco- 
sidal and  other  substances  which  are  quite  readily  decomposed  or  altered 
by  light  and  heat,  will  be  treated  under  ampuls. 

2.  Preparation  of  Ampuls 

Ampuls  (Lat.  ampulla;  Fr.  ampoule; — a  flask)  are  small  glass  con- 
tainers filled  with  medicinal  substances  usually  in  solution.  These  have 
come  into  great  prominence  within  recent  years,  due  to  the  methods  of 
sterilization  now  required  and  practised  in  well  regulated  pharmacies. 
Ampules  are  really  nothing  more  than  very  small  flasks,  the  size  being 
suited  to  single  doses  of  the  medicine,  as  a  rule.  They  were  introduced 
into  France  about  thirty  years  ago  by  Limousin  and  have  now  come  into 
general  use  in  France,  Italy,  Spain,  Holland  and  England.  It  is  only 
recently  that  they  have  come  into  use  in  the  United  States.  C.  A.  Mayo 
was  among  the  first  American  writers  to  publish  the  first  more  complete 
information  regarding  their  origin,  manufacture  and  use.  (See  Proc. 
A.  Ph.  A.,  vol.  57,  1909.)  They  are  generally  adopted  by  the  navies  and 
armies  of  all  civilized  countries,  because  of  the  advantage  which  they  offer 
for  the  preservation,  storage  and  transportation  of  all  manner  of  medicines, 
particularly  those  which  require  sterilization  and  which  are  generally 
wanted  for  immediate  administration.  From  the  standpoint  of  the  physi- 
cian they  are  wonderfully  convenient  and  are  great  time  savers. 


STERILIZATION  AND   DISINFECTION  IN  THE   PHARMACY 


363 


Ampuls  may  have  any  desired  capacity,  from  i  cc.  up  to  100  cc.,  and 
more,  but  the  more  usual  capacities  are  i  cc.,  2  cc.,  5  cc.,  and  10  cc. 
They  are  made  of  alkali-free  glass,  white  or  colored  (amber).  Those  sup- 
plied by  French,  German  and  Italian  makers  are  of  different  forms,  as 
flask-like,  bulb-like,  spindling,  globose,  etc. 


FIG.  86. — Making  ampuls,  a,  Piece  of  glass  rod  to  make  two  ampuls;  &,  rod  a 
heated  in  the  middle  and  nearly  drawn  apart;  c,  d,  two  half  ampuls  filled;  e,  f,  the  com- 
pleted ampuls. 

The  following  are  some  of  the  reasons  why  ampuls  have  come  into  use : 

a.  Most  of  the  liquid  medicamenta  and  those  which  are  to  be  dissolved 
before  using,  have  little  or  no  antiseptic  power  and  under  the  usual  condi- 
tions readily  become  highly  contaminated  with  different  organisms.     The 
use  of  such  contaminated  medicines  has  led  to  serious  infections. 

b.  The  necessity  of  direct  administration  of  medicinal  solutions,  by 


364  PHARMACEUTICAL  BACTERIOLOGY 

hypodermic,  intramuscular  and  intravenous  injection,  is  due  to  the 
desirability  of  getting  prompt  therapeutic  effects. 

c.  The  direct  (hypodermic,  intramuscular  and  intravenous)  adminis- 
tration of  medicamenta  is  very  frequently  necessary  because  administra- 
tion per  mouth  is  impossible. 

As  a  rule  the  pharmacist  will  purchase  ampuls,  ready  for  immediate  use 
by  the  physician,  from  some  reliable  wholesale  manufacturing  house.  In 
certain  districts  and  under  certain  conditions  this  may  not  always  be 
possible,  in  which  case  the  pharmacist  must  prepare  the  ampuls.  The 
pharmacist  should  be  prepared  to  make  all  ampuls  which  may  be  desired 
by  the  Mhysicians  in  his  community.  The  following  suggestions  can  be 
carried  out  readily: 

A.  Glass  Tubing. — Ampuls  can  readily  be  made  from  ordinary  alkali- 
free  glass  tubing,  selecting  rods  of  a  diameter  to  make  ampuls  of  i  cc.,  2  cc., 
5  cc.,  and  10  cc.  capacity.    This  tubing  can  be  secured  from  any  chemical 
or  pharmaceutical  supply  house.     Select  rods  which  are  quite  free  from 
bubbles  and  of  fairly  uniform  diameter  and  thickness. 

B.  Breaking  the  Tubing  into  Suitable  Lengths. — Break  the  tubing  in 
lengths  of  from  five  to  six  inches,  by  filing  a  scratch  with  a  small  file  and 
breaking,  with  the  hands  protected  by  gloves  to  avoid  injury  by  small  bits 
of  glass. 

C.  Sterilizing  and  Neutralizing  the  Glass  Tubing. — Place  the  lengths  of 
class  rods  into  water  with  5  per  cent,  of  soda  and  boil  for  thirty  minutes. 
Neutralize  in  5  per  cent,  hydrochloric  acid,  rinse  thoroughly  and  again 
boil  in  distilled  water.     Let  drain  until  dry.     May  be  placed  in  hot-air 
sterilizer  at  140°  C. 

D.  Making  the  half  Ampul. — Take  one  glass  tube  and  heat  the  middle 
part  in  a  bunsen  burner  with  rotation  until  red  hot  and  soft,  and  pull  apart 
with  a  fairly  quick  strong  pull.     Break  off  the  thin  hairlike  ends  and  hold 
the  tips  in  the  fame  to  seal  them  securely.     A  small  bead  should  form  as 
shown  in  Fig.  86,  c,d,e,f .     A  little  practice  with  a  steady  hand  is  neces- 
sary to  do  this  neatly.     The  half  ampuls  (one  end  open,  the  other  sealed  as 
explained—  are  now  laid  aside  in  a  sterile  box  or  other  container,  until 
ready  to  be  filled.     Or  the  two  ends  of  the  ampul  can  be  reduced  to  a 
capillary  tube  as  follows.     Heat  the  glass  tubing  in  the  blow-pipe  flame, 
beginning  at  one  end,  until  soft  and  draw  out  a  short  distance  with  a  firm 
pull.     Heat  at  a  point  about  i  to  3  inches  from  the  narrowing  portion  of 
the  glass  tube  and  repeat  as  before.     Repeat  this  until  there  are  a  series  of 
tubes  of  normal  diameter  with  capillary  connections.     Breaking  these 
apart  with  the  aid  of  a  file,  yields  empty  ampuls  open  at  the  two  capillary 
ends. 

E.  Filling  the  Half  Ampuls. — This  can  be  done  by  means  of  a  burette, 


STERILIZATION  AND   DISINFECTION   IN   THE   PHARMACY          365 

a  pipette  or  a  medicine  dropper.  The  burette  has  many  advantages. 
Many  ampuls  can  be  filled  from  one  burette,  the  exact  amounts  can  easily 
be  measured.  The  pipette  is  far  less  convenient  than  the  burette  and  is 
more  easily  contaminated.  A  well  graduated  medicine  dropper  is  very 
convenient,  but  all  things  considered  the  burette  is  recommended. 
The  points  to  be  kept  in  mind  are. 

a.  The  finished  ampul  should  not  be  more  than  three-quarters  full. 
The  length  (of  untapered  portion  of  tube)  of  a  neat  ooking  ampul  is  about 
three  or  four  times  the  diameter  of  the  tubing  used. 

b.  In  filling,  introduce  at  least  10  per  cent,  more  than  the  actual  dose 
required,  that  is,  the  i  cc.  tube  should  contain  i.io  cc.;  the  5  cc.  tube 
should  contain  5.50  cc.  of  the  medicinal  substance,  etc.     This  is  to  make 
sure  that  the  physician  may  get  a  full  i  cc.,  5  cc.,  etc.,  dose  after  allowing 
for  unavoidable  loss  (portion  clinging  to  inside  of  ampul,  remaining  in 
narrowed  ends,  etc.). 

c.  In  filling  do  not  allow  any  of  the  liquid  to  come  in  contact  with  the 
upper  end  (open  end)  of  the  tube  as  that  might  interfere  with  sealing. 

There  are  many  different  methods  for  filling  ampuls  which  may  be 
classed  under  three  heads;  filling  by  gravity,  by  pressure,  and  by  vacuum; 
the  latter  two  being  but  modifications  of  the  same  principle  involved. 
There  are  on  the  market  (France,  Holland,  Germany)  several  devices 
made  expressly  for  filling  and  sealing  ampuls. 

F.  Sealing  the  Filled  Half  Ampuls. — This  is  done  by  means  of  suitable 
side-flame  blow-pipe  burner,  pinching  together  and  drawing  our  the  soft  end 
of  the  glass  by  means  of  pincers  and  sealing  in  same  manner  as  the  other 
end.     Do  not  upend  the  ampul  until  it  is  cool,  to  avoid  cracking  the  glass. 

G.  Sterilizing  the  Ampuls. — The   hypodermic   and   other   solutions 
usually  put  up  in  ampuls  can  be  divided  into  three  classes  or  groups  accord- 
ing to  the  degree  of  heat  which  may  or  must  be  used  in  sterilizing,  namely, 
those  which  cannot  withstand  a  temperature  above  60°  C.,  those  which 
can  be  sterilized  at  100°  C.,  and  those  which  may  be  sterilized  in  an 
autoclave  at  120°  C.    Inasmuch  as  the  autoclave  is  rarely  usable  and  also 
because  the  ordinary  steam  temperature  (100°  C.)  will  meet  all  of  the 
requirements  of  the  autoclave,  the  latter  piece  of  apparatus  may  be  left 
out  of  consideration  by  the  practising  pharmacist. 

To  bring  about  a  complete  sterilization  of  the  ampuls,  the  discontinued 
or  fractional  method  should  in  all  cases  be  carried  out.  Place  the  ampuls 
in  a  container  (beaker,  tumbler,  etc.)  with  water  to  which  enough  methyl 
blue  or  f  uchsin  has  been  added  to  give  it  a  very  marked  color  and  sterilize 
as  follows:  If  a  temperature  of  60° C.  is  to  be  used, apply  this  temperature 
(in  incubator  with  Reichert  thermo  regulator)  for  one  hour  each  day  for 
four  to  eight  days.  Some  manufacturers  recommend  a  period  of  ten  days. 
If  the  100°  C.  is  to  be  used,  apply  this  temperature  (in  an  ordnary  Arnold 


366 


PHARMACEUTICAL  BACTERIOLOGY 


steam  sterilizer)  for  from  20  to  30  minutes  once  each  day  for  three  days. 
Should  the  autoclave  be  used,  one  exposure  for  a  period  of  20  minutes  at 
i20°C.  is  sufficient  to  kill  all  organisms,  including  spore. 

It  is  of  vital  importance  in  preparing  liquids  for  hypodermic  and  intra- 
venous injection  to  have  absolutely  perfect  solutions.  There  must  be  no 
insoluble  particles  as  these  might  cause  serious  harm.  After  the  solutions 
are  made  they  should  be  forced  through  a  Berkefeld  or  Pasteur-Chamber- 
land  filter.  All  operations  should  be  done  under  aseptic  conditions,  using 
only  chemically  pure  materials  and  boiled  distilled  water.  If  the  contents 
of  the  ampuls  become  cloudy  after  sterilization,  or  if  the  inside  of  the  glass 
tubes  show  opacities,  something  is  wrong  and  such  ampuls  should  be 
rejected.  Also  reject  all  "leaks,"  indicated  by  the  aniline  color  which 
will  appear  on  the  inside  of  the  tube. 

The  finished  ampuls  are  now  ready  for  use.  The  physician  simply 
breaks  of!  one  end  of  the  ampul,  inserts  the  hypodermic  needle  (sterilized) 
upends  the  ampul  and  aspirates  the  contents  of  the  ampul  into  the  syringe 
by  simply  drawing  down  the  piston.  A  second  method  is  to  remove  the 
piston  from  the  syringe  tube,  break  off  one  end  of  the  ampul,  insert  this  end 
into  the  open  of  the  piston  tube,  break  off  the  other  end  of  the  ampul, 
whereupon  the  contents  will  flow  into  the  piston  tube;  afterward  replace 
the  piston  rod.  In  this  latter  method  great  care  must  be  observed  so  as 
not  to  get  small  particles  of  broken  glass  into  the  hypodermic  syringe. 

Use  white  glass  for  making  ampuls.  Those  filled  with  solutions  which 
are  affected  by  light  may  be  kept  in  an  amber-colored  bottle  or  other  con- 
tainer which  is  impervious  to  light. 

The  following  substances  are  commonly  put  up  in  ampuls.  Many 
others  can  be  so  put  up.  Each  ampul  should  contain  enough  material  for 
one  dose  or  for  one  application,  as  the  case  may  be.  In  the  columns  to  the 
right  are  given  the  sterilization  temperatures;  the  preferred  or  only  usable 
temperatures  being  given  in  degrees,  the  permissible  method  being  indi- 
cated by  "Yes"  and  the  inadmissible  method  being  indicated  by  "No." 
In  case  of  doubt  it  is  always  advisable  to  use  the  lower  temperature  (60°  C., 
hourly  for  from  four  to  eight  days). 


Name  of  Article 

Sterilizingg  Temperature 

Incubator 
60°  C. 

Steam 
100°  C. 

Steam  (auto- 
clave) I20°C. 

Adrenalin  .' 

Yes 
6o°C. 
6o°C. 
6o°C. 
Yes 
Yes 
Yes 

ioo°C. 
Yes? 
No 
Yes? 
ioo°C. 
ioo°C. 
ioo°C. 

No 
No 
No 
No 

No 
No 
No 

Alkaloids  salts  generally  

Antitoxins                                   

Arsenate  of  iron  

Arsenic  

STERILIZATION  AND   DISINFECTION   IN  THE   PHARMACY        367 


Name  of  Article 

Sterilizing  Temperatures 

Incubator 
60°  C. 

Steam 
100°  C. 

Steam  (auto- 
clave) 120°  C. 

Atoxyl                                            

60°  C. 
60°  C. 
60°  C. 
60°  C. 
Yes 
60°  C. 
Yes 
Yes 
Yes 
60°  C. 
60°  C. 
60°  C. 
60°  C. 
60°  C. 
Yes 
60°  C. 
60°  C. 
Yes 
Yes 
60°  C. 
60°  C. 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
60°  C. 
60°  C. 
Yes 
60°  C. 
60°  C. 
Yes 
Yes 
Yes 
60°  C. 
60°  C. 
60°  C. 
60°  C. 

No 
Yes? 
No 
No 
ioo°C. 
No 
100°  C. 
100°  C. 
100°  C. 
No 
No 
No 
No 
No 
100°  C. 
No 
No 
100°  C. 
100°  C. 
No 
No 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
No 
No 
100°  C. 
No 
No 
100°  C. 
100°  C. 
100°  C. 
No 
No 
No 
No 

No 
No 
No 
No 
No 
No 
No 
Yes 
Yes? 
No 
No 
No 
No 
No 
No 
No 
No 
No? 
No 
No 
No 
No 
No 
Yes 
Yes 
No 
No 
No 
Yes 
Yes 
No 
No 
No 
No 
No 
No 
No 
No 
No 
No 
No 
No 
No 

Atropin                                    

Bacterins         

Cacodylates                                 

Caffeine                        

Caffeine  benzoate                                    

Calomel  cream                       

Camphorated  oil  

Chemicals  in  solution                    

Cocaine                     

Duboisine                                                      

Ergot                                            

E&erine  sulphate.       

Eucaine                                        

Gelatin                                    

Olucosid.es                                                             •  . 

Glycerophosphates              

Grey  oil  

Gums                                              

Hysocine                             .            

Iron  cacodylate                                                     •  . 

Mercury  benzoate                           

Mercury  cacodylate 

Mercury  salicylate 

Mercury  sozo-iodolate.        

Mercurial  salts  generally 

Morphine.  .  .           

Mucilaginous  substances 

Normal  salt  solution.  .               

Oils  

Paraffins                                           ... 

Quinine 

Physostigmine. 

Salvarsan  

Scopalamine 

Sera  

Sodium  cacodylate. 

Stovaine 

Strophantin 

Strychnine 

Toxins  

Trypsin. 

Vaccines  

368  PHARMACEUTICAL  BACTERIOLOGY 

Empty  ampuls  of  German  and  French  make  can  be  secured  from 
dealers  in  glass  ware  and  chemical  supplies,  likewise  the  appliances  for 
filling  and  sealing.  These  ready-made  empty  and  filled  ampuls  vary  in 
form  as  already  indicated.  Those  with  a  flat  bottom  and  which  will 
remain  standing  when  placed  on  a  flat  surface  are  preferred  by  some 
physicians. 

The  ready-made  empty  ampuls  (still  sealed)  may  be  sterilized  by  boil- 
ing for  fifteen  minutes  in  a  5  per  cent,  solution  of  phenol,  rinsing  thoroughly 
in  boiling  hot  sterile  water,  draining  and  drying.  With  the  aid  of  a  small 
sharp  file,  break  off  the  tips  of  the  ampuls  to  be  filled.  Place  them  in  dis- 
tilled water,  bring  to  a  boil,  take  vessel  from  the  fire  for  a  few  moments, 
pour  cold  distilled  water  upon  the  empty  floating  ampuls,  a  partial  vacuum 
is  produced  in  the  interior  of  the  ampuls  and  they  quickly  fill  with  water. 
Now  boil  for  thirty  minutes.  When  water  is  sufficiently  cool  take  out  the 
ampuls,  shake  out  the  water  and  dry  in  the  hot-air  sterilizer  at  100°  C. 
They  are  then  ready  to  be  filled,  sealed  and  finally  sterilized  in  the  manner 
already  described.  An  ordinary  sterilized  hypodermic  syringe  will  be 
found  very  satisfactory  for  filling  the  ampuls.  The  suggestions  regarding 
the  amount  of  material  to  be  placed  in  the  ampul,  sealing,  sterilization, 
use  of  the  aniline  solution,  etc.,  already  given,  also  apply  here. 


CHAPTER  XVII 

COMMUNICABLE  DISEASES  WITH  SUGGESTIONS  ON  PRE- 
VENTIVE MEDICINE 

The  pharmacist  should  be  prepared  to  assist  the  physician  and  the 
health  authorities  in  the  enforcement  of  the  sanitary  rules  and  regulations. 
To  this  end  he  should  be  informed  as  regards  the  source  of  the  more  im- 
portant contagious  and  infectious  diseases  and  the  causes  of  epidemics  and 
the  means  available  to  prevent  or  to  combat  such  conditions.  This  does 
not  mean  that  the  pharmacist  must  have  a  full  knowledge  of  the  pathology 


ss 


FIG.  87. — Bacillus  botulinus.  This  bacillus  causes  botulism,  a  form  of  meat  poison- 
ing. There  are  numerous  cases  of  poisoning  resulting  from  eating  infected  meats. 
It  should  be  kept  in  mind,  however,  that  meat  may  not  be  decomposed  and  may  be 
without  bacilli  and  yet  ptomaines  may  be  present.  Therefore  absence  of  bacilli  and 
of  bad  odor  does  not  prove  that  the  meat  is  wholesome.  Meat  from  animals  recently 
killed,  which  has  been  well  cared  for  and  which  is  without  bad  odor  and  shows  no  bacilli, 
is  in  all  probability  wholesome.  Ham,  canned  meats,  cold  storage  meats,  etc.,  may  have 
taken  up  toxins  from  contaminated  meats,  thus  being  made  unfit  for  consumption  even 
though  no  bacteria  are  found. 

and  therapeutics  of  disease.  He  should  have  at  least  a  general  knowledge 
of  the  causes  of  disease  in  order  that  he  may  assist  in  applying  the  means 
for  preventing  disease.  It  is  not  within" the  province  of  the  pharmacist  to 
cure  disease,  but  he  should  be  a  potent  factor  in  preventive  medicine. 

In  many  instances,  protection  against  one  kind  of  infection  also  pro- 
tects against  other  infections.     About  1893,  two  health  officers  (H.  F. 
Mills  of  the  Massachusetts  State  Board  of  Health  and  Dr.  J.  J.  Reinke 
24  369 


370  PHARMACEUTICAL  BACTERIOLOGY 

of  the  Hamburg  city  Board  of  Health)  noted  that  a  reduction  in  deaths 
from  typhoid  fever,  due  to  improved  water  sanitation,  coincided  with 
a  reduction  in  mortality  from  causes  other  than  typhoid.  This  is  known 
as  the  Mills-Reinke  phenomenon  and  has  received  much  attention  on  the 
part  of  health  officers  everywhere.  This  observation  is  however  not  so 
recent  as  is  indicated  by  the  date  given.  For  many  years,  it  has  been 
general  knowledge  among  observing  health  officers  and  practicing  physi- 
cians, that  an  improvement  of  those  conditions  which  reduce  the  mortality 
rate  due  to  any  one  of  the  more  or  less  serious  intestinal  infections,  reduces 
the  general  mortality  rate  likewise,  and  it  has  been  surmised  for  many 
years  that  some  kind  of  genetic  relationship  exists  between  all  filth  borne 
pathogens  and  toxigens.  It  makes  little  difference  against  which  of  the 
many  filth  germs  the  sanitary  activities  of  a  city  or  community  are  pri- 
marily directed,  the  end  result  will  be  a  reduction  in  all  filth  borne  diseases. 
A  thorough  and  complete  sanitary  cleanup  of  a  city  means  a  reduction  in 
the  following  diseases,  naming  them  in  the  order  of  their  more  usual 
pathogenetic  filth  relationship.  The  entire  series  of  intestinal  infections, 
as  the  diarrheas,  dysenteries,  ententes,  colitis,  cholera  infantum,  cholera 
morbus,  etc.;  typhoid  fever,  tuberculosis  (including  both  the  bovine  and 
human  types),  diptheria,  and  incidentally  also  syphilis  and  gonorrhea. 
The  coincident  reduction  in  the  so-called  social  diseases  is  no  doubt  due 
to  the  fact  that  a  purely  physical  cleaning  up,  encourages  or  stimulates 
moral  cleanliness.  To  the  list  of  essentially  filth  borne  diseases  must  be 
added  Asiatic  cholera,  amebic  dysentery,  bubonic  plague,  and  other 
diseases  endemic  in  certain  countries.  The  exact  causality  of  some  of  the 
supposedly  water  borne  diseases,  such  as  goiter,  has  as  yet  not  been  fully 
worked  out. 

As  early  as  1886-1887,  the  writer  was  stronly  impressed  by  the  high 
rate  of  intestinal  diseases  in  the  city  of  Chicago  and  surmised  so  some 
causal  relationship  between  the  dysenteries  and  typhoid.  Since  the 
completion  of  the  Chicago  canal,  the  general  health  of  that  city  has  im- 
proved wonderfully.  Up  to  that  time,  the  sewage  of  the  city  was  dumped 
into  lake  Michigan  (via  the  Chicago  river)  and  again  pumped  into  the  city 
and  the  inhabitants  were  obliged  to  swallow  the  mixed  human  and  animal 
excreta.  The  accumulation  of  the  sewage  in  the  sluggish  stream  gave 
rise  to  an  undescribable  stench,  still  fresh  in  the  memories  of  the  older 
members  of  the  present  generation  of  the  city.  The  digging  of  the  sani- 
tary canal  and  the  proper  diverting  of  the  city  sewage,  is  the  grandest  and 
best  thing  ever  done  by  the  city  of  Chicago. 

A  contagious  disease  is  one  which  is  readily  communicable,  from  one 
person  or  animal  to  another,  either  through  direct  contact  or  very  close 
proximity.  An  infectious  disease  is  communicable  through  a  considerable 


COMMUNICABLE   DISEASES  371 

interval  of  space.  Itch,  for  example,  is  contagious,  but  not  in  the  least 
infectious,  whereas  whooping-cough  is  infectious,  but  not  contagious. 
Some  diseases  are  both  contagious  and  infectious,  as  small-pox  and  diph- 
theria. Malaria  and  yellow  fever  are  infectious,  but  not  in  the  least  con 
tagious.  Since  the  distinction  between  contagious  and  infectious  diseases 
cannot  be  clearly  drawn,  these  term  are  discontinued  and  the  term  com- 
municable diseases  is  substituted  therefor.  By  communicable  disease 
we  mean  any  disease  which  may  be  transferred  from  the  sick  to  the  well, 
either  directly  through  close  contact  or  indirectly  through  more  or  less 


PIG.  88. — Bacillus  anihracis.  This  bacillus  is  spore-forming  and  causes  the  cattle 
disease  known  as  anthrax.  This  disease  is  especially  common  among  sheep  and  cattle 
and  may  be  transmitted  to  man,  especially  those  working  with  the  wool,  hides  and 
meat  oi  infected  animals.  The  two  chief  forms  of  anthrax  in  man  are  malignant  pustule 
and  woolsorter's  disease.  The  dried  spores  of  this  bacillus^  will  live  for  years  and  will 
withstand  the  boiling  temperature  for  hours.  Vaccinating"animals  against  anthrax  is 
commonly  practised  now.  Anthrax  is  frequently  confused  with  glanders,  an  equine 
disease  caused  by  the  Bacillus  mallei,  a,  Non-spore-bearing  bacilli;  &,  chains  of  cells;  c, 
spore-bearing  bacilli.  Cell-walls  and  plasmic  contents  are  stained,  the  spores  are  un- 
stained. 

distance  in  space.  A  pandemical  disease  is  one  which  spreads  over  or 
pervades  the  entire  earth.  La  Grippe  or  influenza  is  such  a  disease.  The 
last  pandemic  destroyed  more  lives  than  were  killed  during  the  entire 
period  of  the  World  War,  and  this  despite  the  fact  that  the  mortality  rate 
in  this  disease  is  not  high.  The  case  rate  was  very  high,  about  equal  in 
numbers  to  the  entire  armies  engaged  in  the  World  War.  In  the  past  Asi- 
atic cholera  and  plague  have  been  pandemical  in  scope.  In  modern  times 
pandemics  are  prevented  by  the  national  quarantine  services,  and  any 
extensive  epidemic  in  any  country  or  state  is  prevented  by  the  state  and 
community  quarantine.  That  is,  this  is  certainly  true  as  far  as  the  disease 
are  concerned  concerning  which  we  know  the  cause.  The  only  reason 
why  influenza  became  pandemical  is  because  we  are  riot  as  yet  aware  of 
the  primary  cause  A  disease  which  is  more  or  less  wide  spread  over  a 
country  is  spoken  off  as  an  epidemic.  For  example,  cerebrospinal  mening- 
itis and  pneumonia  may  be  epidemical.  Diphtheria  is  often  epidemic  in  a 
community,  and  as  above  stated,  it  is  likewise  infectious  and  contagious. 


372 


PHARMACEUTICAL  BACTERIOLOGY 


The  term  epidemic  is,  however,  also  applied  to  any  communicable  disease 
which  has  become  general  in  a  given  community.  A  more  or  less  common 
or  spreading  disease  which  is  limited  to  and  recurs  in  a  given  district  or 
country  is  said  to  be  endemic  in  that  district  or  country.  Endemics  are 
usually  due  to  climatic  conditions  which  encourage  certain  microbic  and 
other  disease-producing  invasions. 

The  causes  of  disease  are  of  two  kinds,  primary  or  inciting,  and  second- 
ary or  predisposing.  The  primary  cause  of  a  disease  is  that  factor  or  in- 
fluence which  must  invariably  be  active  before  the  disease  can  possibly 
develop.  For  example,  the  primary  cause  of  diphtheria  is  the  diphtheria 
bacillus;  the  predisposing  causes  are  exposure  to  .wet  and  cold,  impover- 
ished condition  of  body,  etc.  No  matter  how  numerous  or  how  active  the 
predisposing  causes  may  be,  the  disease  cannot  develop  until  the  primary 
cause  acts.  There  are  numerous  abnormal  or  pathological  states  or  con- 
ditions without  recognizable  primary  causes,  as  gout,  rheumatism  and  the 
senile  changes  in  the  body;  and  again  there  are  certain  diseases  which 
evidently  have  primary  causes,  as  whooping-cough,  small-pox  and  yellow 
fever,  but  in  which  said  primary  causes  are  not  yet  discovered.  The 
following  tabulation  outlines  the  primary  and  secondary  causes  of  disease: 

Bacteria,  as  in  typhoid  and  Asiatic  cholera. 

Protozoa,  as  in  malaria. 

Primary  causes  Parasitic  higher  animals,   as  tape-worm    and 

(inciting).  itch. 

Fungi,  as  in  ring-worm  and  pellagra. 

Undetermined,  as  in  small-pox. 


Communicable 
diseases. 


Secondary  causes 
(predisposing) . 


Heredity . . 


Age. 


Sex. 


Environment. 


Family. 

Individual  (ontogenetic). 


Infancy. 
Childhood. 
Adolesence. 
Adult. 
Old  age. 


Climate. 

Altitude. 

Seasons. 

Unsuitable  food. 

Unsuitable  clothing. 

Poisons. 

Occupation. 


Habits. 


Injuries. 

Alcoholic. 
Tobacco. 
Drugs. 

Coffee  and  tea. 
1  Gourmandage. 


COMMUNICABLE   DISEASES  373 

In  a  general  way  it  may  be  stated  that  any  cause,  factor  or  influence, 
which  tends  to  lower  the  vitality,  predisposes  to  disease.  Individuals  with 
a  well-balanced  physical  and  mental  development  are  less  liable  to  disease, 
and  when  attacked  are  more  apt  to  recover,  than  those  individuals  who 
have  a  poor  physical  development.  Undue  abstinence  is  as  harmful  as 
over-indulgence.  The  ascetic  is  as  pathologic  as  the  gouty  gourmand. 


FIG.  89.  FIG.  90. 

FIG.  89. — Bacillus  mallei,  the  cause  of  glanders  in  horses.  This  disease  can  be  trans- 
mitted to  man  where  it  causes  symptoms  of  a  suppurative  infection  of  the  lymphatic 
glands.  Mallein,  which  is  used  in  testing  horses  for  glanders,  consists  of  the  filtrate 
(Berkeield  filter)  of  dead  cultures  (glycerin  bouillon)  of  the  bacillus.  A  positive  malleiu 
reaction  consists  in  a  rise  in  temperature  and  local  swelling.  The  dose  is  i  c.c. 

FIG.  90. — Bacillus  tetani,  an  anaerobic  spore-bearing  bacillus,  the  cause  of  tetanus  or 
lockjaw.  This  bacillus  is  found  in  soils  and  may  infect  abrasions,  cuts  and  wounds. 
Treatment  with  tetanic  antitoxin  is  successful  if  begun  before  the  symptoms  develop. 
The  best  time  to  administer  the  antitoxin  is  at  the  time  the  injury  is  received. 


FIG.  91. — A  spore-bearing  bacillus  stained  with  methyl  blue  leaving  the  spores  un- 
stained.    Fortunately  most  of  the  bacilli  pathogenic  to  man  do  not  bear  spores. 

Irrational  diet,  drink  and  food  fads,  sooner  or  later  leave  their  pernicious 
effects  upon  the  system  and  predispose  to  certain  diseases.  Overeating 
is  as  objectionable  as  starvation.  Lack  of  adequate  physical  exercise  has 
its  evil  effects  as  does  also  over-exertion.  Trained  or  professional  athletes 
are  not  long  lived,  many  are  hopelessly  afflicted  with  enlarged  and  weak- 
ened heart  and  arteries  (aneurism).  Pernicious  habits  of  all  kinds  indicate 


374  PHARMACEUTICAL  BACTERIOLOGY 

weakness  and  further  develop  the  weakness,  which  in  turn  predisposes  to 
certain  diseases  and  render  the  individual  less  resistant  to  the  ravages  of 
disease.  A  good  ancestry  and  inheritance,  good  wholesome  food,  comfort- 
able clothing,  the  right  sort  of  exercise  for  body  and  mind,  the  simple  life 
rather  than  the  strenuous  life,  avoiding  bad  habits  of  all  kinds,  abundant 
fresh  air,  etc.,  all  tend  toward  longevity.  To  argue  that  we  should  go  un- 
clothed is  as  absurd  and  unreasonable  as  to  teach  that  sheep  should  be 
shaved.  To  adhere  to  a  wholly  vegetable  diet  is  irrational  simply  because 
we  are  organically  adapted  to  a  mixed  diet.  An  excessive  meat  diet  is  also 
very  pernicious. 

Occupation  is  a  potent  factor  in  predisposing  to  disease,  and  in  lon- 
gevity. The  following  table  adapted  from  a  report  by  Ogle  will  serve  to 
make  this  clear.  The  high  mortality  rate  among  street-hawkers  is  due  to 
several  causes  chief  of  which  are  low-living,  exposure  to  inclement  weather, 
and  the  greater  exposure,  in  the  squalid  districts  of  large  cities,  to  the 
primary  causes  of  disease.  The  low  mortality  rate  among  clergymen  is 
due  to  a  comparatively  simple  though  comfortable  mode  of  living;  while  in 
the  case  of  the  farmer  and  gardener,  the  out-of-door  life  is  the  favorable 
influence.  The  list  represents  ages  ranging  from  twenty-five  to  sixty 
years,  therefore  adults. 

Occupation  Comparative 

Mortality 

Clergymen,  priests  and  ministers TOO 

Gardeners 108 

Farmers 114 

Carpenters 14? 

Lawyers 152 

Coal  miners 160 

Bakers 172 

Builders,  masons,  bricklayers 174 

Blacksmiths 175 

Commercial  clerks 1 79 

Tailors 189 

Cotton  manufacturers 196 

Medical  men 202 

Stone,  slate  quarries 202 

Book-binders 210 

Butchers 211 

Glass  workers 214 

Plumbers,  painters,  glaziers 216 

Cutler,  scissors  makers 229 

Brewers 245 

Innkeeper,  liquor  dealers 274 

File  makers 300 

Earthenware  workers 3*4 

Street  hawkers 33& 

Inn,  hotel  service 396 


COMMUNICABLE   DISEASES  375 

The  following  are  the  more  important  communicable  diseases  with 
suggestions  on  prevention.  The  information  is  given  for  the  sole  purpose 
to  better  qualify  the  pharmacist  to  cooperate  with  the  health  officers  in 
safeguarding  the  public  health. 

A.  Tuberculosis. — Commonly  known  as  consumption  and  the  "  white 
plague."  A  universal  disease,  essentially  infectious,  especially  peculiar  to 
crowded  habitations  and  to  lack  of  pure  fresh  air.  The  primary  cause  is 
the  Bacillus  tuberculosis  (bacillus  of  Koch),  a  non-spore-bearing  microbe, 
which  is  somewhat  more  resistant  to  disinfectants  and  other  destructive 
agencies  than  most  other  pathogenic  bacteria.  The  chief  predisposing 
causes  are  living  in  crowded  habitations 
and  inherited  low  vitality,  especially  weak 
lungs.  The  disease  may  be  general  (gen- 
eral tubercular  infection)  or  it  may  be 
localized  in  any  one  or  in  several  organs 
or  tissues.  Commonly  localized  in  the 
lungs  (phthisis,  consumption)  and  in 
lymph  glands.  Lupus  and  many  so-called 
scrofulous  conditions  are  tuberculosis  of  il %  ^ 

the  skin;  the  disease  often  attacks  bones 

j   .    .    ,      /T_.      .    .    ,    j.  r     i  M  i        \  FIG.  92. — Bacillus  tuberculosis. 

and  joints  (hip-joint  disease  of  children).    Although  this  organism  does  not 


It  attacks  young  and  old  and  may  OCCUr    form  spores  it  is  quite  resistant  to 
ii         11         r  i-r          mr      j«  •        the    action    of    germicides.      The 

in  all  walks  of  life.  The  disease  enters  ma  bacillus  causing  the  bovine  type 
the  air  passages  and  per  mouth  with  food  °f  tuberculosis  differs  slightly  in 
and  drink.  It  is  contracted  by  inhalation  ££&£,  3hS£&±£.  ** 
through  close  association  with  consump- 
tives, and  the  bovine  form  or  type  of  tuberculosis  is  acquired  from  the 
milk  of  tubercular  cows.  Bovine  tuberculosis  is  especially  liable  to  affect 
the  lymph  glands  and  the  joints  in  children,  rarely  in  adults. 

The  disease  sometimes  runs  a  quick  course  (quick  consumption),  but 
more  generally  it  makes  an  insidious  start  and  runs  a  chronic  course. 
Many  people  have  limited  local  infections  which  are  only  discovered  at  an 
autopsy.  There  are  many  spontaneous  recoveries  from  tuberculosis. 
Since  it  is  very  important  to  begin  early  treatment,  the  physician  resorts  to 
several  tests  for  the  purpose  of  determining  the  possible  existence  of 
masked  or  incipient  forms  of  the  disease.  These  tests  are  as  follows  and 
depend  upon  the  reactions  produced  by  tuberculins  when  applied  to  or 
introduced  into  the  system: 

a.  The  Calmette  or  Ophthalmo  Test. — Old  tuberculin,  precipitated  by 
alcohol  is  used.  The  precipitate  is  dried  and  made  into  a  i  per  cent,  solu- 
tion in  sterilized  distilled  water  or  sterile  physiologic  salt  solution.  This 
substance  is  put  up  in  sterile  capillary  pipettes,  ready  for  use.  A  drop  of 


376  PHARMACEUTICAL  BACTERIOLOGY 

the  solution  is  placed  in  one  eye,  using  the  other  eye  as  a  control.  Any 
abnormality  in  the  eye  is  regarded  as  a  contraindication.  If  tuberculosis 
exists  in  the  system  it  is  indicated  by  an  inflammation  in  the  eye  tested. 
Also  known  as  the  Wolff-Eisner  test  or  reaction.  It  may  be  necessary  to 
repeat  the  test  several  times  before  satisfactory  results  are  obtained. 

b.  The  wnPirquet  or  Cutaneous  Test. — A  25  per  cent,  solution  of  tuber- 
culin (O.  T.)  is  applied  to  the  skin  with  scarification,  as  in  vaccination. 
The  skin  is  first  cleansed  with  alcohol  and  control  scarifications  are  made 
near  the  test  area.    This  test  is  also  known  as  the  "skin  reaction."     It  is 
not  very  reliable.     The  inflammatory  reaction  may  be  simulated  by  other 
substances  in  persons  that  are  known  to  be  entirely  free  from  tuberculosis. 

c.  The  Moro,  Percutaneous  or  Ointment  Test. — Fifty  per  cent,  tuberculin 
(O.T.)  in  lanolin  is  rubbed  into  the  skin,  without  scarification.     The  prep- 
aration is  put  up  in  collapsible  tubes,  one  tube  containing  enough  material 
for  several  tests.     If  tuberculosis  exists,  small  reddened  vesicles  appear  at 
the  point  of  inunction,  usually  on  the  second  day. 

d.  The  Thermal  Test. — A  solution  of  tuberculin  (O.T.)  put  up  in, 
8  cc.  bottles,  representing  one  milligram  per  cc.  (i-iooo)  is  injected  hypo- 
dermically.     If  tuberculosis  is  present  there  is  a  rise  in  temperature, 
usually  within  ten  to  twenty-four  hours  after  injection. 

e.  The  Detre  Differential  Test.— This  test  is  intended  to  differentiate 
between  tuberculosis  of  human  origin  and  that  of  bovine  origin.    Three 
tuberculins  are  required.    Tuberculin  O.  T.,  tuberculin  B.  F.,  made  from 
tubercle  bacilli  of  human  origin  and  tuberculin  B.  F.,  made  from  tubercle 
bacilli  of  bovine  origin.    Three  small  skin  areas  are  scarified.    Into  one 
tuberculin  O.  T.  is  rubbed,  into  the  second  humanized  tuberculin;  and  into 
the  third  bovinized  tuberculin.     The  resulting  reactions  indicate  whether 
tuberculosis  is  of  human  or  of  bovine  origin. 

We  cannot  go  into  the  details  of  the  reactions.  They  are  not  always 
reliable,  neither  the  positive  nor  the  negative  reactions.  In  the  advanced 
stages  of  tuberculosis  and  in  moribund  cases,  the  reaction  is  usually  nega- 
tive. Indeed,  in  such  cases  the  test  is  unnecessary  as  the  existence  of  the 
disease  is  evident  without  special  tests. 

Tuberculosis  is  not  as  infectious  as  is  generally  supposed.  Those  who 
are  in  good  condition  physically  may  live  for  years  with  those  afflicted  with 
the  disease  without  becoming  infected.  Yet,  tubercular  patients  should 
be  isolated  from  well  people  as  much  as  possible.  The  sputum  is  the 
principal  source  of  infection,  also  other  secretions;  and  the  breath  as  in 
sneezing,  laughing  and  coughing.  Plenty  of  fresh  pure  dry  air  should  be 
supplied  to  patients,  large  airy  sleeping  rooms  and  easily  digested  whole- 
some food  is  essential.  Consumptives  should  not  marry,  should  not 
kiss  healthy  individuals,  especially  children.  Expectorated  material 


COMMUNICABLE   DISEASES  377 

should  be  disinfected  at  once.  Treatment  should  be  begun  early.  The 
propaganda  favoring  well  constructed,  well  ventilated,  comfortably 
warmed  homes  and  less  close  segregation  in  cities  and  a  general  improve- 
ment in  sanitation  will  do  much  toward  eradicating  tuberculosis.  Tene- 
ment houses  and  large  or  small  crowded  houses  of  all  kinds  should  not  be 
tolerated  for  moral  as  well  as  for  sanitary  reasons.  The  milk  used  must 
be  free  from  tubercular  infection.  Dry  warm  climates  and  the  higher 
clear  atmospheres  favor  recovery  from  tuberculosis  (California,  Colorado). 
The  following  test  has  been  found  very  reliable  in  the  hands  of 
clinicians. 

The  Jefimoff -Klein  Urinary  Test  for  Tuberculosis 

1.  Mix  4  cc.  of  the  fresh  urine  with  2  cc.  of  a  20  per  cent,  lead 
acetate  solution,  warm  and  filter  several  times  while  hot. 

2.  The  filtrate,  while  still  hot,  is  mixed  with  ten  or  more  drops 
of  an  alcoholic  solution  of  silver  nitrate.     A  precipitate  forms  which 
varies  in  color  from  brick  red  to  cherry  red,  according  to  the  progress 
of  the  disease.     Normal  urine  remains  uncolored  (white  precipitate). 

The  silver  nitrate  solution  is  made  by  dissolving  10  grams  of  silver 
nitrate  in  a  minimum  of  water  and  adding  strong  alcohol  up  to 
100  cc. 

Most  tuberculous  urine  is  amphoteric  (amphochromatic) ,  when 
warmed,  that  is  it  will  turn  red  litmus  blue,  and  blue  litmus  red. 
This  behavior  may  be  utilized  as  a  corroborative  test  with  the  above. 
In  advanced  stages  of  the  disease  the  urine  becomes  markedly  acid, 
and  is  no  longer  amphoteric. 

B.  Typhoid  Fever. — This  is  a  filth  disease.  If  the  environment  were 
made  clean  and  sanitary,  typhoid  fever  could  not  exist.  The  primary 
cause  is  the  non-sporulating  Bacillus  typhosus  which  is  found  in  filthy  water, 
in  milk  and  in  food  materials.  Typhoid  contaminated  slops,  sewage, 
wash  water,  etc.,  poured  on  the  soil  may  seep  into  the  well  water  and  finally 
enter  the  system  in  drinking.  The  bacillus  develops  readily  in  the  intestinal 
tract  where  the  reaction  is  alkaline.  It  is  quite  susceptible  to  the  action 
of  weak  acids  and  is  easily  killed  by  boiling  and  by  disinfectants.  Ty- 
phoid is  a  widely  disseminated  dangerous  infectious  as  well  as  contagious 
disease.  In  large  cities  the  mortality  rate  from  this  disease  is  directly 
proportional  to  the  filthiness  of  the  drinking-water  supply.  In  country 
districts  epidemics  are  very  frequently  due  to  contaminated  well-water 
(contaminated  from  kitchen  refuse,  barns,  cow-sheds,  etc.).  Epidemics 
often  follow  in  the  wake  of  the  dairyman,  who  supplies  cow's  milk  in  cans 
washed  with  or  which  contain  milk,  contaminated  with  polluted  water. 
Typhoid  fever  is  carried  in  vegetables  from  truck  gardens  where  human 
and  other  excrement  are  used  for  fertilizing  purposes.  The  Chinese 


378  PHARMACEUTICAL  BACTERIOLOGY 

truck  gardeners  are  particularly  culpable  in  this  regard.  Again,  the 
vegetables  may  be  irrigated  with  stagnant  sewage-polluted  water.  House 
flies  are  carriers  of  typhoid. 

The  mortality  rate  in  typhoid  is  high  and  the  disease  runs  its  course  in 
about  five  weeks.  There  are  some  mild  cases,  the  so-called  walking  or 
ambulatory  cases.  All  of  the  excreta  from  the  patient  should  be  disin- 
fected. Among  the  disinfectants  which  have  been  used  for  this  purpose 
are,  corrosive  sublimate  (i-iooo),  copper  sulphate  (5  to  15  per  cent.), 
copperas  solution  (10  to  20  per  cent).  These  are  effective  when  properly 
used.  Their  albumen  coagulating  coefficient  is  very  high  (see  table  in  the 
chapter  on  disinfectants)  and  it  is  absolutely  necessary  to  stir  the  mixture 
of  material  and  disinfecting  solution  very  thoroughly.  The  noncoagulat- 
ing  disinfectants  are  to  be  preferred,  such  as  milk  of  lime  and  the  coal  tar 
disinfectants.  All  bed  linen,  clothing,  etc.,  used  by  the  patient  should 
be  disinfected  in  5  per  cent,  carbolic  acid  before  washing.  Everything 
used  by  the  patient  should  be  sterilized,  disinfected  and  kept  away  from 
the  rest  of  the  family.  Those  who  nurse  typhoid  patients  must  be  ex- 
tremely careful  not  to  carry  the  infection  to  others.  Pillows,  mattresses 
and  other  large  articles  used  by  the  patient  should  be  steam  sterilized. 
In  simple  words,  everything  about  the  patient  must  be  scrupulously  steri- 
lized in  order  to  avoid  spreading  the  infection. 

A  national  department  of  health  should  see  to  it  that  the  water  supply 
of  large  cities  is  free  from  sewage  contamination.  Our  streams,  lakes  and 
reservoirs  which  supply  drinking  water,  require  careful  guarding  against 
typhoid  infection. 

There  should  be  compulsory  regulation  regarding  the  position  and 
depth  of  wells  in  farm  yards  and  as  regards  the  position  of  the  well  relative 
to  barns,  cow  sheds,  privy  vaults,  etc.  Typhoid  fever  will  continue  its 
ravages  as  long  as  filth  contamination  of  water  supplies  and  food  supplies 
is  permitted. 

The  Gruber-Widal  test  for  typhoid  is  an  agglutination  phenomenon. 
The  agglutinating  power  of  the  blood  of  a  typhoid  patient  is  usually 
noticeable  as  early  as  the  fifth  day  of  the  disease.  Preventive  inoculation 
with  typhoid  bacterin  has  been  used  with  considerable  success,  particu- 
larly in  the  British  and  German  armies,  ane  is  now  quite  extensively  used 
in  general  practice.  Chantamesse  and  Wright  use  agar  or  broth  cul- 
tures of  the  typhoid  bacillus,  killed  by  heat. 

The  de  Silvestri  urinary  test  for  typhoid  is  made  as  follows: 

In  a  small  test  tube  overlay  2   mils  of  ferric  chloride  to  which 

four  drops  of  concentrated  sulphuric  acid  has  been  added,  with  three 

mils   of   the   filtered   urine.     If   typhoid   is   present,  a   more   or   less 

maroon  colored  ring  will  develop  at  the  zone  of  contact,  and  at  the 


COMMUNICABLE   DISEASES  379 

top  of  the  upper  layer  a  turbid  ring  exhibiting  a  green  fluorescence 
develops.  The  reaction  is  said  to  be  most  distinct  with  urine  from 
persons  suffering  with  paratyphoid  A,  but  also  with  that  of  persons 
suffering  with  typhoid  and  paratyphoid  B. 

The  decade  just  passed  has  proved  to  the  entire  world  that  typhoid 
fever  is  one  of  the  readily  preventable  diseases.  This  is  done  by  means 
of  the  bacterins  (ordinary  and  sensitized).  As  the  result  of  the  use  of 
these  agents  typhoid  fever  has  been  driven  from  the  army.  The  bacterins 
establish  immunization  which  is  an  efficient  as  the  vaccination  against 
mallpox.  It  now  lies  within  the  means  of  everyone  to  protect  himself 
agaihst  this  disease.  (See  also  the  army  statistics  mentioned  under 
serobacterins) . 

For  some  time  the  human  typhoid  carriers  (that  is,  persons  who  har- 
bored the  typhoid  organism  without  showing  signs  of  the  disease)  have 
received  much  attention  and  numerous  cases 
have  been  traced  to  such  carriers.     The  at-  ^    o         Q 

tempts  to  free  such  carriers  from  the  infecting  ^    o  ^  ^      &  ^ 

organisms    have  as    a    rule,   not    met   with  ^  ^       «=»^  ^  °  <=> 

general  success.  0  ^X     ^^^ 

C.  Pneumonia. — Pneumonia      with      its      <^^^Q     0  <?   0    ^ 
modifications,  as  broncho-pneumonia,  capil-   0    0^      ^ Q  ^ 

lary  bronchitis,  pleuro-pneumonia,  pneumonic     .  ^    ^     ^     ^0      ^ 
pericarditis,  etc.,  is  estremely  common.     The  ^    ^       ^ 

primary    cause   of   pneumonia  is   the    Dip- 

FIG.  93. — Bacillus  pneumonias 
loCOCCUS     pneumomae    of    which    three    types     Of   Friedlander,  also  known  as 

(type  I,  type  II  and  type  III)  and  one  group    Bacillus  mucosus.    This  organ- 

.  or-     jsm  ls  non_Sporogeneous  and  is 

(group  IV)  are  recognized.     Types  I  and  II    easily  killed, 
cause  about  33  per  cent,  of  all  cases.     Type 

II  occurs  in  about  10  to  15  per  cent,  of  cases  which  are  very 
severe  with  a  morality  rate  of  50  per  cent.  The  group  IV 
form  the  causative  organisms  in  about  45  to  50  per  cent,  of  cases, 
having  about  the  same  mortality  rate  as  for  groups  I  and  II,  namely 
10  to  15  per  cent  .  The  important  predisposing  causes  are  exposure  to 
wet  and  cold,  weak  lungs,  infancy,  old  age,  general  debility  and  alcoholism. 
It  is  generally  limited  to  the  respiratory  tract  and  the  contiguous  tissues, 
as  the  pericardium  and  the  pleurae.  Among  infants  and  young  children 
and  those  well  past  middle  life,  the  disease  shows  a  high  mortality  rate. 
In  youth  and  early  middle  life  recovery  is  the  rule,  provided  the  physical 
inheritance  and  development  is  good.  The  mortality  rate  among  those 
addicted  to  the  use  of  alcoholic  drinks,  and  those  affected  with  "tobacco 
heart,"  is  very  high. 

One  attack  of  pneumonia  is  supposed  to  increase  the  resisting  power 


380  PHARMACEUTICAL  BACTERIOLOGY 

to  subsequent  attacks  but  such  acquired  immunity  is  not  by  any  means 
permanent.  The  anti-pneumococcic  serum  is  used  with  some  apparent 
success  though  the  results  are  far  from  satisfying  to  the  majority  of  those 
who  have  tried  it.  Dr.  Shafer  has  recently  recommended  a  mixed  bac- 
terin  (composed  of  disease  exudate  and  pure  pneumococcic  bacterin)  which 
has  been  used  with  some  success.  At  the  present  time  the  use  of  the 
specific  bacterin  (ordinary  and  sensitized)  is  much  used  as  a  cure,  employing 
the  polyvalent  bacterin  in  those  cases  where  diagnosis  is  uncertain,  and 
in  cases  where  it  is  not  convenient  or  possible  to  make  the  differential 
tests.  Three  differential  tests  are  recommended,  the  urine  test,  the 
sputum  test  and  the  exudate  test.  Confirmatory  cultural  tests  and  micro- 
scopical examinations  are  also  made.  The  details  of  the  differential  as 
well  as  the  confirmatory  tests  cannot  be  given  here.  They  may  be  found 
in  the  larger  more  comprehensive  manuals  of  medical  bacteriology. 

It  is  important  to  guard  against  exposure  to  wet  and  cold,  particularly 
when  the  vitality  of  the  body  is  lowered,  as  through  lack  of  sleep,  lack  of 
food,  over-exertion,  etc.  The  sputa  of  patients  should  be  disinfected  at 
once.  Well  persons  having  good  resisting  power  may  carry  the  germs 
and  convey  the  disease  to  those  who  have  a  lower  vitality.  The  room 
occupied  by  the  patient  should  be  thoroughly  fumigated  as  soon  as  possible. 

D.  Small-pox. — Also  known  as  variola  and  pest.  This  is  a  well- 
known  disease  which  has  occurred  epidemically  from  time  to  time  through- 
out all  ages  and  in  all  lands.  It  is  most  highly  infectious  and  contagious. 
In  spite  of  all  investigations,  the  primary  cause  has  not  yet  been  discovered. 
The  contagion  is  wafted  from  the  skin  eruptions  and  is  carried  in  clothing 
and  by  everything  used  or  touched  by  the  patient.  The  contagion  may 
lie  dormant  in  clothing  for  months.  The  contagion  is  known  to  be 
filterable,  will  remain  active  in  glycerin  for  months,  will  resist  drying  for 
weeks  but  is  quickly  rendered  inert  by  bile  and  by  sodium  oleate.  Heat- 
ing for  15  minutes  at  a  temperature  of  58°  C.  will  also  destroy  it.  Certain 
cell  inclusions  (infected  epithelial  cells)  have  been  considered  the  primary 
causative  organisms,  and  have  been  named  Cytoryctes  vaccinae.  These 
are  very  minute  but  may  readily  be  seen  in  the  epithelial  debris  of  the 
vaccine. 

All  excreta  from  the  patient  should  be  disinfected  with  a  5  per  cent, 
solution  of  carbolic  acid  or  other  convenient  disinfecting  agents  as  lime, 
formalin,  etc.  Bedding,  mattresses  and  other  material  used  in  the  sick- 
room should  be  burned  as  soon  as  the  patient  does  not  need  them  any 
longer. 

As  the  result  of  the  general  practice  of  vaccination  (with  the  modified 
cow  virus)  small-pox  is  no  longer  the  dread  disease  that  it  once  was. 
In  Germany,  where  smallpox  vaccination  has  been  religiously  enforced 


COMMUNICABLE   DISEASES  381 

from  the  first,  this  disease  is  practically  non-existent.  In  fact  so  rare,  is 
the  disease  that  the  medical  society  of  Berlin  some  years  ago,  elected  one 
member  each  year  who  shall  give  a  lecture  on  this  disease  in  order  that 
the  fraternity  might  retain  some  idea  as  to  the  nature  of  this  disease.  In 
Austria,  in  France  and  in  the  United  States  where  anti-smallpox  vaccina- 
tion is  less  rigidly  enforced  the  case  rate  is  occasionally  high,  and  lesser 
epidemics  appear  from  time  to  time,  with  varying  mortality  rates.  One 
minor  epidemical  outbreak  in  Berkeley  (California)  of  the  confluent 
hemorrhagic  type  of  the  disease  gave  a  mortality  rate  of  100  per  cent. 
Since  vaccination  is  almost  an  absolute  safeguard,  there  is  no  need  of  fear- 
ing this  disease,  even  when  brought  in  direct  contact  with  it.  One  vaccina- 
tion does  not  always  establish  life  immunity,  as  is  popularly  believed. 
The  rule  is  to  vaccinate  in  infancy,  again  about  the  time  of  adolescence 
and  aeain  in  early  adult  life.  This  will  usually  insure  immunity  for  life. 
However,  vaccination  should  be  carried  out  after  every  exposure  or  when- 
ever smallpox  exists  in  the  vicinity,  no  matter  how  many  good  "take" 
scars  there  may  be.  Nurses  and  physicians  in  pest  hospitals  are  vacci- 
nated once  a  year,  or  oftener,  to  insure  immunity.  In  the  navy  it  is 
customary  to  vaccinate  every  man  every  time  a  port  is  entered  where 
small-pox  is  suspected.  Small-pox  is  a  quarantinable  disease. 

There  is  absolutely  no  danger  or  ill  effects  from  vaccination,  in  spite 
of  the  popular  newspaper  and  popular  verbal  reports  to  the  contrary.  In 
perhaps  one  case  in  a  million,  tetanus  or  severe  septicaemia  may  be  traceable 
to  the  use  of  an  impure  virus.  Septic  infection  of  the  scarified  area  may 
take  place,  due  to  carelessness  on  the  part  of  the  patient,  and  not  due  to  the 
virus  used,  but  even  this  is  an  extremely  rare  occurrence.  Since  the 
incubation  period  of  small-pox  is  about  twelve  days  and  that  of  vaccinia 
(cow-pox)  is  only  five  or  six  days,  it  is  evident  that  the  vaccination  will 
establish  immunity  even  in  those  who  were  actually  exposed,  provided 
vaccination  is  done  within  a  few  days  after  exposure. 

Primitive  (savage)  races  are  very  susceptible  to  small-pox,  with  a 
very  high  mortality  rate.  This  is  in  part  due  to  the  total  ignorance 
of  sanitary  measures,  resulting  in  the  more  ready  spread  of  the  contagion. 
Entire  savage  tribes  have  been  exterminated  by  this  disease.  Negroes 
are  far  more  susceptible  than  Caucasians.  Indians  have  spread  the  infec- 
tion in  blankets  after  having  been  exposed. 

E.  Malaria. — This  familiar  disease,  commonly  known  as  ague,  the 
shakes,  chills  and  fever,  and  intermittent  fever,  prevails  in  many  areas  in 
the  United  States  and  is  limited  to  swampy  wet  countries.  It  gradually 
disappears  with  the  tilling  and  the  draining  of  soil  which  remove  the  breed- 
ing places  of  the  only  carriers  of  the  disease,  namely  the  mosquitos  (An- 
opheles). The  primary  causes  is  the  Plasmodium  malaria  (Hamatozoa 


382  PHARMACEUTICAL  BACTERIOLOGY 

malaria)  which  is  introduced  into  the  circulation  by  the  sting  of  the  mos- 
quito. 

The  prophylactic  measures  consist  in  the  destruction  of  the  mosquitos 
in  rooms.  To  this  end  burn  two  pounds  of  Pyrethrum  to  every  thousand 
cubic  feet  of  space.  Sulphur  (one  pound  per  thousand  cubic  feet)  may  be 
used  though  it  offers  no  advantage  over  the  Pyrethrum  and  has  the  dis- 
advantage of  corroding  metal  and  fading  colored  fabrics.  Also  destroy 
the  breeding  places  of  the  mosquito  and  keep  mosquitos  out  of  houses 
by  means  of  screens  and  netting.  Protect  the  person  against  mosquito 
stings  when  travelling  in  countries  known  to  be  infested  by  the  Anopheles 
group  of  mosquito.  Also  take  quinine  as  a  prophylactic  (3  to  5  grains 
twice  daily),  and  as  a  cure.  Quinine  is,  however,  more  satisfactory  as  a 
preventive  than  as  a  cure. 

The  life  history  of  the  malarial  plasmodium  is  complex  but  it  has  been 
worked  out  very  definitely.  Two  life  cycles  are  recognized,  the  asexual 
(also  known  as  the  human  cycle,  the  cycle  of  Golgi  and  the  schizogonic 
cycle),  the  sexual  (also  known  as  the  mosquito  cycle,  cycle  of  Ross  and 
sporogonic  cycle),  and  occasionally  a  third  cycle  known  as  the  partheno- 
genetic  or  virgin  cycle,  which  is  said  to  explain  the  latent  occurrences 
of  the  organism  in  the  human  body.  The  spindle  shaped  sporozoites 
resulting  from  the  sexual  generation  in  the  mosquito  are  introduced  into 
the  human  body  by  the  sting  of  the  female  member  of  the  Anopheline 
group  of  mosquitoes  (male  mosquitoes  do  not  bite).  The  leucocytes  of 
the  blood  devour  as  many  of  these  sporozoites  as  they  can.  Those  not 
so  destroyed,  enter  the  red  blood  corpuscles  where  they  undergo  the  signet 
ring  stage  (socalled,  because  the  stained  specimens  show  a  resemblance 
to  a  signet  ring)  of  development,  finally  greatly  enlarging  and  partially 
disintegrating  the  blood  corpuscle  by  their  increase  in  size  and  numbers, 
forming  the  merocyte.  The  matured  merocytes  divide  into  a  number 
of  small  bodies  which  occur  free  in  the  blood  plasm,  constituting  the 
merozoites.  This  cycle  in  the  human  blood  is  completed  in  from  twenty- 
four  to  seventy-two  hours,  depending  upon  the  species  of  malarial  organ- 
ism. Each  merozoite  now  enters  a  red  blood  corpuscle  and  a  similar  cycle 
repeats  itself.  At  each  sporulation  a  paroxysm  of  fever  manifests  itself. 
It  was  found  that  most  of  the  merozoites  were  asexual,  but  that  some  were 
sexual.  The  sexual  forms  require  longer  time  to  mature  (from  eight  to 
ten  days)  and  are  known  as  the  gametocytes.  From  the  sporozoite  intro- 
duced by  the  sting  of  the  mosquito  to  fully  matured  gametocytes  (male 
and  female)  constitutes  the  complete  asexual  or  human  cycle.  The 
gametes  are  now  ready  to  enter  upon  the  sexual  cycle  which  can  take 
place  only  in  the  salivary  glands  of  the  female  member  of  the  Anopheline 
group  of  mosquitos.  The  mosquito  inoculates  itself  with  gametocytes 


COMMUNICABLE   DISEASES  383 

upon  filling  itself  with  blood  of  a  malarial  patient  in  whom  the  asexual 
cycle  has  been  completed.  Should  the  mosquito  bite  the  human  earlier, 
that  is,  before  the  gametes  are  matured,  it  would  not  become  a  transmitter 
of  malaria  for  obvious  reasons.  In  the  sexual  or  mosquito  cycle,  the 
gametocytes  derived  from  the  human  undergo  the  preliminary  change 
in  the  stomach  of  the  insect  (flagellation  and  exflagellation  of  the 
male  gametocyte  and  the  macrogamete  formation  of  the  female  gameto- 
cyte).  The  products  of  exflagellation  constitute  the  male  gametes  or 
microgametes  which  now  fertilize  the  macrogametes,  giving  rise  to  the 
ookinetes.  The  oogonites  now  pass  through  the  wall  of  the  stomach 
and  attach  themselves  to  or  lie  adjacent  to  the  outer  lining  of  the  stomach, 
where  they  grow  to  large  size  forming  cysts  in  which  are  developed  hun- 
dreds of  tiny  spindle  shaped  nucleated  bodies  known  as  the  sporozoites, 
which  enter  the  body  cavity  of  the  mosquito  from  which  they  gradually 
are  gathered  into  the  salivary  glands,  where  they  remain  until  some  of 
them  may  be  injected  with  the  saliva  into  the  human  body,  where  the 
asexual  cycle  again  repeats  itself. 

Malaria  could  be  made  to  disappear  from  the  face  of  the  earth  by  doing 
the  following. 

1.  Destroy  all  malaria  bearing  mosquitos,  or, 

2.  Destroy  all  humans,  or, 

3.  Do  away  with  the  breeding  places  of  malaria  bearing  mosquitos 
occurring  within   the  zones  of   dissemination  for  humans,   comprising 
what  is  commonly  known  as  malarial  control. 

Since  the  malaria  lorganism  cannot  survive  unless  it  is  provided  with 
the  two  hosts,  namely  man  and  mosquito,  it  is  evident  that  the  discon- 
tinuance of  one  or  the  other  of  the  two  hosts,  would  cause  the  malaria 
to  disappear.  Proposition  (i)  is  practically  impossible  and  proposition 
(2)  is  not  to  be  thought  of.  Proposition  (3)  is  practicable  as  has  been  dem- 
onstrated on  numerous  occasions.  It  is  reasonable  to  suppose  that  if 
all  breeding  places  of  the  malaria  spreading  mosquitos  occurring  within 
the  reach  of  humans  should  be  rendered  uninhabitable  for  the  mosquito 
larvae,  for  a  period  sufficiently  long  to  make  sure  that  all  latent  carriers 
were  also  dead,  then  the  disease  would  be  eradicated  from  the  face  of 
the  earth. 

Malarial  control  is  a  very  definite  phase  of  sanitary  science  and  those 
who  are  interested  should  consult  the  following  special  treatise.  Herms, 
WilJiam,  B. — Malaria:  Cause  and  Control.  The  Mac  Millian  Company. 

F.  Diphtheria. — This  dread  disease  is  both  infectious  and  contagious. 
The  primary  cause  is  the  Bacillus  diphtheria,  also  known  as  the  Klebs- 
Loeffler  bacillus.  The  chief  predisposing  causes  are  exposure  to  wet  and 
cold.  The  disease  may  be  localized  in  the  larynx  (membranous  croup)- 


384  PHARMACEUTICAL  BACTERIOLOGY 

in  the  pharynx,  in  the  nares,  on  any  of  the  mucous  membranes,  and  in 
cuts  and  wounds.  Animals  such  as  cats  and  dogs  may  carry  the  infection. 
The  sick  must  be  isolated  and  all  discharges  from  nose,  mouth  and  throat 
as  well  as  the  bed  linen,  etc.,  must  be  sterilized  and  disinfected.  Upon 
recovery,  the  sick-room  must  be  thoroughly  fumigated  by  means  of  for- 
maldehyde. Bedding,  mattress  and  pillows  must  be  disinfected.  The 
anti-diphtheric  serum  should  be  used  early  and  in  large  doses.  The  best 
authorities  look  upon  this  remedy  as  a  specific  always  effecting  a  cure, 
provided  it  is  given  in  time  and  given  in  adequate  doses.  All  those  who 
have  been  exposed  should  receive  a  proplylactic  dose  of  the  remedy  (about 
1,000  units).  The  other  remedial  agents  as  gargles,  sprays,  etc.,  should 
not  be  neglected.  The  diphtheria  toxin  acts  on  the  heart  and  all  patients 
should  be  warned  against  any  sudden  or  severe  exertion  until  complete 
recovery  is  assured  by  the  attending  physician  as  death  has  resulted  from 
a  single  undue  action,  as  jumping  or  suddenly  rising  from  bed. 

G.  Cancer. — The  piimary  cause,  the  secondary  causes  and  the  treat- 
ment of  cancer  are  all  in  the  dark  as  yet.  We  know  that  this  disease  rarely 
develops  eailier  than  middle  life.  It  usually  runs  a  comparatively  short 
course  (several  months  to  two  years),  producing  some  rather  marked 
symptoms  (the  cancerous  cachexia),  with  constant  pain,  and  a  very  char- 
acteristic waxy  pallor  of  the  skin.  It  is  to  be  hoped  that  the  primary 
cause  and  the  cure  will  be  discovered  in  a  short  time.  There  are  some  indi- 
cations that  a  tendency  to  cancer  is  inherited  and  that  the  primary  cause 
is  an  organism  resembling  the  protozoa  group.  There  is  a  popular  belief 
that  eating  raw  tomatoes  causes  cancer,  and  it  may  be  that  the  plasmod- 
ium  of  cancer  resides  in  some  vegetable.  Cancer  may  attack  any  tissue 
or  organ,  although  the  internal  viscera,  as  liver  and  stomach,  are  more  com- 
monly affected.  Cancer  should  be  treated  as  a  contagious  disease  though 
the  proof  of  its  contagious  nature  is  not  conclusive. 

All  advertised  cancer  cures  are  fakes.  There  is  no  known  cure  for 
cancer.  Surgical  removal  of  cancerous  growths  has  been  the  means  of 
prolonging  life,  but  the  trouble  is  very  apt  to  recur.  Many  cases  are 
inoperable. 

Among  the  agencies  which  have  been  tried  as  cures  for  cancer  are 
mixed  streptococcic  bacterins;  radium  emanations  from  various  sources, 
as  radium  and  other  radioactive  minerals,  water  rendered  radioactive,  and 
radioactive  clay;  and  plant  extracts.  None  of  them  have  proved  satisfac- 
tory. Radium  has  undoubtedly  effected  some  cures  and  improvements 
but  it  is  far  from  reliable.  The  principle  upon  which  the  use  of  radium 
emanations  are  based  are  as  follows.  The  emanations  are  destructive 
to  livingT tissues,  but  even  more  destructive  to  pathological  tissues.  A 
carefully^adjusted  dose  of  emanations  will  kill  pathological  tissue,  such  as 


COMMUNICABLE   DISEASES  385 

cancer,  without  seriously  injuring  normal  tissues.  In  skin  cancer  the 
properly  adjusted  dosage  and  time  exposure  of  the  radium  emanations 
has  resulted  in  the  complete  killing  of  the  cancerous  epithelium,  so  that 
it  could  be  lifted  off,  showing  the  normal  granulation  tissue  underneath. 
Many  volumes  have  been  written  about  cancer  and  many  scientific 
bodies  have  given  much  time  and  attention  to  the  cancer  problem,  which 
is  a  very  serious  one,  but  the  solution  is  not  yet  found. 

H.  Plague.  —  This  disease,  which  is  also  known  as  black  plague,  the 
pest,  bubonic  plague,  black  death,  etc.,  is  essentially  a  filth  disease.  The 
primary  cause  is  the  non-sporogenous  Bacillus  pestis.  The  plague  has 


FIG.  94.  PIG.  95. 

PIG.  94.  —  Bacillus  pestis.  Does  not  form  spores  and  is  very  easily  killed.  The  ends 
stain  more  heavily  than  the  middle.  Involution  forms  may  occur.  Sometimes  the 
cells  become  encapsuled  as  shown  in  the  figure. 

FIG.  95.  —  Bacillus  cholera  also  known  as  Spirillum  cholera,  the  case  of  Asiatic 
cholera. 

occurred  epidemically  from  time  to  time  throughout  all  ages.  It  is  most 
virulent  and  most  prevalent  (endemic)  in  the  crowded  cities  of  the  warmer 
countries  (Oriental  cities),  where  the  sanitary  conditions  are  often  very 
bad.  The  disease  is  highly  contagious  and  infectious  and  is  communicable 
not  only  to  man  but  also  to  rats,  mice,  dogs,  squirrels  and  cattle.  Rats, 
and  the  fleas  upon  -them,  are  the  principal  carriers  of  the  disease,  although 
other  animals,  as  ants  and  flies,  may  also  act  as  carriers. 

There  are  several  forms  of  the  plague  of  which  the  pneumonic  is  the 
most  dangerous  and  most  infectious  because  the  bacilli  are  spread  by 
coughing  and  sneezing. 

In  this  disease  thorough  disinfection  is  of  the  greatest  importance. 
The  entire  body  of  the  patient  should  be  washed  with  a  disinfecting  solu- 
tion (1-1200  bichloride  of  mercury).  Disinfect  everything  used  about  the 
patient.  After  death  or  recovery  everything  used  by  the  patient  should 
be  destroyed  by  burning. 
25 


386  PHARMACEUTICAL  BACTERIOLOGY 

Rats  (bearing  the  infected  fleas)  are  the  principal  carriers  of  this 
disease,  and  the  experience  in  San  Francisco  (1906-1909)  has  demonstrated 
that  plague  disappears  as  soon  as  the  plague  infested  rats  disappear.  De- 
stroy rats  and  mice  and  see  to  it  that  the  home  is  free  from  fleas.  Plague 
is  a  quarantinable  disease  and  the  federal  authorities  are  constantly  on 
the  lookout  to  prevent  the  importation  of  this  disease.  The  Oriental 
ports  are  the  chief  sources  of  infection. 

Yersin's  anti-plague  serum  and  Haffkine's  bacterin  have  been  used  with 
considerable  success  as  prophylactics  and  also  with  some  success  as  cures. 

I.  Asiatic  Cholera. — This  is  another  filth  disease  essentially  of  Oriental 
origin,  particulaly  prevalent  in  the  crowded  unsanitary  cities  of  India 
and  Asia.  It  is  a  quarantinable  disease.  The  primary  cause  is  the  non- 
sporogenous  Bacillus  cholera  (Spirillum  cholera)  also  known  as  the 
comma  bacillus  of  Koch.  The  principal  sources  of  the  infection  are  pol- 
luted water  and  food,  particularly  the  former.  In  fact  the  sources  of  in- 
fection and  modes  of  entry  into  the  digestive  tract  are  not  unlike  those 
of  typhoid.  Cholera  is  highly  infectious  and  usually  occurs  epidemically, 
often  spreading  over  wide  areas.  Human  excrement  carries  the  infection 
and  when  this  material  is  used  as  fertilizer,  which  is  done  in  China  and 
other  Oriental  countries,  it  becomes  the  means  of  initiating  and  continuing 
the  spread  of  the  disease.  The  importing  by  the  Chinese  of  human  ex- 
crement and  animal  dung  for  medicinal  purpose  should  be  prohibited 
as  it  may  be  the  means  of  starting  an  epidemic  of  cholera  in  the  United 
States,  even  though  it  is  unlikely  that  the  infection  will  survive 
the  trip. 

Fortunately  the  cholera  bacillus  is  easily  killed  by  heat,  disinfectants, 
and  by  drying.  The  temperature  of  boiling  water  kills  it  in  five  minutes. 
In  water  it  may  retain  its  vitality  for  a  long  time.  Furthermore,  it  is  not 
a  strict  (obligative)  parasite  and  may  multiply  outside  of  the  body  under 
favorable  conditions.  Flies  carry  the  infection  from  cholera  stools  to 
articles  of  food. 

Haffkine's  attenuated  cholera  bacterin  has  been  employed  successfully 
as  a  prophylactic.  The  method  of  use  consists  first  in  the  hypodermic  in- 
jection of  a  weak  virus,  that  is,  cultures  attenuated  by  long  cultivation  at  a 
high  temperature  (39°  C).,  and  following  this  later,  in  five  days,  with  a 
virulent  culture.  More  recently  Kolle  has  used  cultures  killed  by  heating 
for  one  hour  at  58°  C.,  which  has  given  good  results  in  numerous  tests  made 
during  a  cholera  epidemic  in  Japan.  Pf  eiffer  and  others  have  experimented 
extensively  with  cholera-immune  serum  and  have  demonstrated  that  this 
has  marked  lyctic  properties.  The  cholera  bacilli  when  placed  into  the 
serum  first  lose  motility,  then  swell  up  into  coccus-like  forms  and  finally 
dissolve.  This  property  is  said  to  be  due  to  two  substances,  one  found  in 


COMMUNICABLE   DISEASES  387 

normal  serum  and  the  other  found  in  immune  serum.  Neither  substance 
alone  can  destroy  the  cholera  bacilli  but  the  two  acting  together  are 
strongly  bacteriolytic.  The  immunity  produced  by  the  Haflkine  and 
Kolle  bacterins  is  temporary  only. 

J.  Yellow  Fever. — This  highly  infectious,  but  in  no  wise  contagious 
disease,  is  peculiar  to  tropical  and  subtropical  countries.  The  primary 
cause  is  as  yet  unknown  but  it  is  supposed  to  be  a  protozoan.  The  sole 
carrier  of  the  infection  is  a  mosquito,  Aedes  calopus.  The  disease  has 
been  highly  epidemical  in  the  southern  states  but  since  the  discovery  of  the 
part  played  by  the  mosquito  the  mortality  rate  has  been  lowered  to  a 
marked  degree.  In  fact  the  disease  is  now  under  complete  control.  No 
Aedes  mosquitos,  no  yellow  fever. 

It  has  been  observed  for  a  long  time  that  a  frost  checked  the  disease  at 
once,  which  as  is  now  known,  was  due  to  the  fact  that  the  frost  killed  the 
carriers  of  the  infection.  In  a  general  way  the  statements  made  under 
malaria  prophylaxis  also  apply  here.  Caucasians,  especially  those  not 
acclimated  in  the  yellow-fever  countries,  are  very  susceptible  to  the  disease; 
Negroes  and  Latin  races  are  far  less  susceptible. 

The  history  of  the  establishment  of  complete  yellow  fever  control  is  of 
intense  interest.  As  early  as  1881  Dr.  Finlay  of  Havana  suggested  that 
the  mosquito  was  responsible  for  yellow  fever,  based  upon  the  obser- 
vation that  the  disease  disappeared  as  soon  as  the  colder  weather  killed 
the  animal.  In  1900  the  United  States  appointed  the  Yellow, Fever  Com- 
mission placed  under  the  direction  of  Dr.  Walter  Reed,  with  James 
Caroll,  Jesse  W.  Lazear  and  Aristides  Agramonte  as  associates.  Of 
these  Lazear  died  of  yellow  fever  and  Caroll  took  the  disease  but  recovered. 
Dr.  Reed  has  since  also  died,  of  spotted  fever,  which  he  contracted  while 
investigating  this  fatal  cattle  disease.  The  commission  was  stationed  at 
Cuba  (Havana)  and  the  outcome  of  their  investigation  may  be  sum- 
marized as  follows. 

1.  The  specific  primary  cause  was  not  found,  but  it  was  supposed  to  be 
an  organism  similar  to  that  which  causes  malaria. 

2.  The  fever  is  transmitted  by  the  bite  of  a  mosquito. 

3.  The  mosquito  must  harbor  the  infecting  agent  about   12   days 
before  it  becomes  actively  transmissible  to  man.     The  earlier  bites  (1-8 
and  10  days)  are  harmless. 

4.  The  mosquito  Aedes  calopus  is  the  intermediary  host  of  the  primary 
cause  of  yellow  fever,  while  man  is  the  definitive  host. 

5.  Yellow  fever  has  an  incubation  period  of  from  41  hours  to  5  days. 

6.  Yellow  fever  is  not  carried  or  spread  by  fomites.     The  use  of  dis- 
infectants is  of  no  avail  against  the  spreading  of  yellow  fever. 

7.  One  attack  of  yellow  fever  establishes  immunity  to  subsequent 
attacks. 


388  PHARMACEUTICAL  BACTERIOLOGY 

8.  Yellow  fever  can  be  prevented  by  destroying  the  specific  carriers 
of  the  disease,  namely  the  Aides  calopus. 

9.  The  Aides  calopus  (formerly  Stegomyia  calopus)  must  feed  upon  a 
yellow  fever  patient  during  the  first  few  days  of  the  disease  in  order 
that  said  mosquito  may  become  a  carrier  of  the  disease. 

Insect  powder  (Pyrethrum)  is  employed  to  destroy  mosquitoes  in 
houses.  Sprays,  crude  oil,  etc.,  are  used  on  ponds,  pools,  and  stagnant 
water  in  yellow  fever  districts  to  destroy  the  mosquito  larvae.  Drainage 
of  wet  lands,  of  swamps,  of  pools,  etc.,  prevents  the  development  of  mos- 
quitoes. Rain  barrels  and  cisterns  may  breed  the  yellow  fever  mosquito. 
Cold  weather  and  frosts  check  yellow  fever  because  the  mosquitoes  are 
killed. 

The  work  of  the  yellow  fever  commission  made  possible  the  digging  of 
the  Panama  canal  and  yellow  fever  epidemics  are  a  thing  of  the  past. 
The  work  of  the  commission  is  far  better  known  and  better  appreciated  in 
Europe  than  it  is  in  the  United  States.  It  should  also  be  mentioned  that 
there  were  many  others  attached  to  the  commission  whose  names  are  not 
generally  mentioned,  as  soldiers,  nurses  and  attendants,  who  are  deserving 
of  much  credit  for  the  success  of  the  remarkable  work  done. 

K.  Pellagra. — Pellagra  is  a  disease  which  has  created  great  havoc  in 
Italy  and  other  Eastern  countries,  and  which  first  appeared  in  the  United 
States  about  1907.  It  spread  very  rapidly  and  up  to  1911  numerous  cases 
have  been  reported  from  the  Southeastern  United  States  and  from  Illi- 
nois, with  a  few  scattering  cases  from  Kansas,  Virginia,  Pennsylvania, 
New  York,  Massachusetts,  California,  and  other  states.  The  disease  is 
said  to  be  caused  by  eating  moldy  corn  (Zea  mays)  or  foods  prepared 
from  such  corn.  Ceni  and  others  declare  that  the  primary  cause  is  a 
species  of  Aspergillus  (A .  flavescens  and  perhaps  also  A .  fumigatus) .  It  is 
also  believed  that  the  ordinary  household  mold  (Penicillium  glaucum) 
is  a  primary  cause.  The  mortality  rate  is  very  high,  and  the  disease  is 
said  to  be  terrible  in  its  effects.  It  first  manifests  itself  as  an  eruption 
of  the  skin  usually  appearing  in  the  early  spring,  February  or  March, 
after  some  variable  prodromal  symptoms.  The  skin  becomes  darkened 
and  blotchy.  Eczematous  eruptions  next  appear,  with  desquamation. 
Gradually,  as  the  older  eruptions  heal,  while  new  ones  form,  the  skin 
becomes  rough,  from  which  the  name,,  pell'  agra — rough  skin — is  derived. 
The  symptoms  increase  from  year  to  year.  The  nervous  manifestations 
are  varied  and  are  accompanied  by  great  suffering. 

Pellagra  is  not  contagious  or  infectious,  though  the  tendency  is  trans- 
mitted from  one  generation  to  another.  Childern  of  pellagrins  are  often 
born  with  asymmetrical  heads  and  various  other  deformities.  They  may 
be  idiotic  or  stupid  and  defective  generally. 


COMMUNICABLE   DISEASES  389 

Acute  pellagra  runs  a  rapid  course,  but  more  generally  it  is  chronic,  the 
suffering  continuing  for  years  in  an  ever  increasing  ratio.  The  sufferers 
simply  degenerate  from  year  to  year  and  die  a  slow  terrible  death. 

Lombrosa,  Ceni  and  others  recognized  the  fact  that  pellagrins  are 
mostly  of  the  poorer  class,  whose  principal  diet  is  polenta,  a  mush  made 
from  corn  meal.  This  much  is  usually  prepared  in  large  potfuls,  sufficient 
for  a  week's  eating,  and  set  away,  exposed  to  dust,  dirt,  flies,  etc.,  so  that 
these  ignorant  peasants  often  eat  polenta  which  is  more  or  less  moldy  and 
otherwise  spoiled.  Efforts  were  at  once  made  to  correct  these  conditions, 
but  proved  only  partially  successful  as  far  as  checking  the  ravages  of  the 
disease  was  concerned.  The  primary  cause  of  pellagra  is  not  yet  dis- 
covered. Dr.  Louis  W.  Sambon  of  the  London  School  for  tropical  medicine 
asserts  that  maize,  either  sound  or  spoilt,  is  not  the  cause  of  the  disease, 
that  it  is  decidedly  endemic  in  its  tendencies,  that  its  stations  are  closely 
associated  with  streams  of  running  water,  and  suggests  that  a  small 
blood  sucking  fly  belonging  to  the  genus  Simulium  is  the  agent  by  which 
pellegra  is  conveyed.  Others  suggest  that  it  is  a  dietary  disease  indicated 
by  the  fact  that  the  disease  does  not  develop  in  those  who  use  a  welJ  mixed 
and  well  balanced  diet. 

L.  Syphilis. — The  primary  cause  of  syphilis  is  the  Treponema  pallidum 
(Spirochaeta  pallida),  belonging  to  the  group  of  protozoa  known  as  the 
Zoomastigophora  (Flagellata) .  The  life  history  is  still  unknown.  The 
full  life  cycle  appears  to  be  far  more  complex  than  was  originally  supposed. 
The  male  organism  is  the  form  usually  recognized  as  the  Treponema  of 
syphilis.  The  female  cell  is  supposed  to  develop  in  certain  body  cells, 
as  the  lymphocytes  and  the  endothelial  cells.  The  idea  of  the  bisexual 
nature  of  the  organism  is  gaining  more  and  more  credence  among  bacterio- 
logists. These  matters  cannot  be  entered  into  in  a  work  of  this  kind. 

Syphilis  as  well  as  gonorrhea  are  filth  diseases  in  the  sense  that  with 
absolute  physical  cleanliness,  as  well  as  moral  cleanliness,  these  diseases 
could  not  exist.  Sanitarians  have  made  the  interesting  observation  that 
cities  and  towns  that  were  subjected  to  a  thorough  cleaning  up  as  a  safe- 
guard against  the  spreading  of  some  infectious  disease,  such  as  cholera  or 
typhoid  fever,  also  showed  a  decrease  in  cases  of  the  so-called  social  diseases. 
It  is  known  that  in  those  establishments  for  prostitutes  where  physical 
cleanliness  is  required  and  strictly  enforced  (primarily  for  business  reasons 
only),  the  case  rate  for  the  two  diseases  is  much  reduced.  While  it  is 
true  that  the  great  majority  of  cases  are  traceable  to  promiscuous  inter- 
course, this  factor  per  se  plays  no  part  in  the  dissemination  of  the  two 
diseases,  excepting  in  so  far  as  this  promiscuity  increases  the  chances 
for  contact  with  physical  uncleanliness  on  the  part  of  both  sexes,  the 
female  in  particular.  A  decrease  or  increase  in  promiscuity  has  no  pri- 


3QO  PHARMACEUTICAL  BACTERIOLOGY 

mary  influence  as  to  any  increase  or  decrease  in  cases.  The  reason  why 
the  morally  clean  are  quite  free  from  the  diseases  is  because  they  do  not 
expose  themselves.  The  immoral  men  and  women  (that  is,  sexually  highly 
promiscuous)  might  be  equally  free  from  infection  provided  they  were 
themselves  physically  clean  and  kept  away  from  those  who  were  infected. 

It  is  essentially  chronic  in  its  course,  the  effects  being  apparent  even 
/n  the  third  and  fourth  generations.  Primitive  races  are  said  to  have  been 
free  from  this  disease  until  the  advent  of  civilization,  yet  the  disease  is  of 
great  antiquity  having  been  widespread  in  ancient  Rome  and  Greece.  It 
is  very  infectious  via  abrasions,  cuts  and  all  breaks  in  the  continuity  of 
the  skin  and  mucous  membranes.  The  infection  is  carried  by  all  manner 
of  exposed  objects,  as  clothing,  dentist's  instruments,  pipes,  dishes,  drinking 
vessels,  etc.,  in  fact  anything  and  everything  which  may  have  been  in 
contact  with  a  syphilitic.  The  primary  lesions  of  the  patients  are  very 
infectious. 

The  disease  is  readily  preventable.  All  that  is  necessary  is  to  keep 
away  from  the  carriers  of  the  infection.  Syphilitics  should  be  isolated 
until  cured.  The  disease  is  very  readily  kept  under  control  by  the  proper 
remedial  agents,  but  persistency  in  the  use  of  medicines  is  necessary 
to  effect  a  cure.  Ehrlich's  606  (Salvarsan),  is  considered  in  the  nature  of 
a  specific,  given  in  hypodermic,  intramuscular  or  intravenous  injections. 

M.  Gonorrhea. — This  is  also  a  filth  disease.  The  primary  cause  is  the 
non-sporogenous  Micrococcus  (Diplococcus)  gonorrhea.  It  is  not  infectious 
but  exceedingly  contagious  to  mucous  membranes.  As  Ophthalmia 
neonatorum  (ophthalmia  of  the  new-born)  it  is  a  very  fruitful  cause  of 
blindness.  The  suppurative  discharges  from  patients  are  highly  con- 
tagious. The  contagion  is  carried  by  patients  and  by  the  articles  touched 
or  handled  by  them.  The  disease  is  difficult  to  eradicate  from  the  system. 
It  is  not  so  frequently  localized  in  urethra  and  vagina  as  is  generally 
supposed,  but  it  may  travel  to  the  bladder,  kidneys,  joints,  etc.,  and  it 
may  be  general  upon  nearly  all  mucous  membranes  of  the  body.  It  is  very 
apt  to  become  chronic,  giving  rise  to  very  serious  after  effects.  Syphilis 
and  gonorrhea  have  the  following  in  common. 

1.  Both  are  highly  contagious  by  direct  contact,  but  particularly  so  to 
mucous  membranes.     They  are  in  no  sense  infectious  and  are  epidemic 
or  general  only  in  proportion  to  the  number  of  contact  inoculations.     The 
chief  carriers  and  disseminators  of  the  contagions  are  the  women  in  public 
houses  and  the  male  frequenters  of  such  houses.    Lack  of  personal  cleanli- 
ness is  a  very  fruitful  source  of  spreading  the  infection. 

2.  The  innocent  (infants,  children  and  adults)  are  occasionally  infected 
through  contact  with  those  afflicted  with  the  diseases,  as  in  shaking  hands, 
kissing,  contact  with  clothing  and  other  articles  used  by  those  already  in- 


COMMUNICABLE   DISEASES 


391 


fected.  Physicians,  dentists,  and  nurses  may  become  accidentally 
infected.  Physicians  and  dentists  may  inoculate  patients  accidentally, 
through  the  use  of  improperly  disinfected  instruments;  this  is,  however, 
quite  rare.  Contaminated  drinking  vessels,  spoons,  forks,  etc.,  may 
transmit  the  infection. 

3.  In  both  diseases  the  primary  causes  are  readily  destroyed  by  the  use 
of  disinfectants.  With  absolute  cleanliness  the  diseases  could  not  exist. 
In  brief,  the  two  diseases  could  not  exist  if  moral  and  physical  cleanliness 
prevailed. 


PiG.  96. — Gonococcus  and  pus  cells  from  theurethral  discharges  of  acute  gonorrhea 
The  organism  is  readily  demonstrated  by  the  usual  staining  methods,  using  monhylene 
blue  or  Gram's  method.  The  Gonococcus  is  cultured  with  some  difficulty  (use  blood 
serum-agar  in  incubator  at  37°C.)«  There  are  several  other  cocci  resembling  the  Gono- 
coccus in  form,  but  these  differ  in  that  they  can  be  cultured  in  ordinary  media  at  the 
room  temperature.  (Williams.) 


4.  Both  diseases  are  difficult  to  cure  as  already  stated.     Both  are  and 
do  become  general  or  systemic  in  character,  and  are  not  local  as  is  generally 
supposed.     Those  suffering  from  these  diseases  should  be  isolated  and 
should  never  be  allowed  to  come  in  close  contact  with  the  innocent. 

5.  Physicians,  pharmacists  and  nurses  should  act  as  public  agents  in 
giving  information  regarding  the  transmissibility  of,  and  the  difficulty  of 
curing  syphilis  and  gonorrhea  and  pointing  to  clandestine  prostitution  as 
the  most  active  source  of  the  contagion.     It  should  be  made  a  criminal 
offense  for  a  syphilitic  to  convey  the  contagion  to  an  innocent  person.     In 
the  army  and  navy  the  men  receive  careful  instruction  as  to  preventive 
measures.     This  was  found  necessary  as  the  prevalence  of  these  diseases 
incapacitated  a  large  percentage  of  the  men  from  active  duty. 


-3Q2  PHARMACEUTICAL  BACTERIOLOGY 

In  the  treatment  and  cure  of  syphilis  mercurial  and  arsenical  prepara- 
tions and  the  iodides  play  a  very  important  part.  In  the  treatment  of 
gonorrhea,  disinfectants,  especially  silver  nitrate  and  protargol,  play  a 
very  important  part.  The  antigonorrheic  bacterin  has  been  used  with 
some  success  as  a  prophylactic  and  as  a  cure  in  chronic  cases.  Only 
competent  physicians  can  treat  these  diseases  properly.  All  advertised 
and  patented  "quick  cure"  remedies  are  fakes. 

Ehrlich  and  Hata  have  discovered  what  appears  to  be  a  specific  in  the 
treatment  of  syphilis,  namely,  intramuscular  and  intravenous  injections 
of  dioxydia-amidoarsenobenzol  (Salvarsan,  or  "No.  606").  The  tests 
thus  far  made  have  yielded  astonishing  results.  Many  of  the  most 
severe  forms  of  the  disease  have  been  promptly  cured  by  a  single  dose  of 
this  remedy. 

The  Wassermann  or  Wassermann-Noguchi  test  for  syphilis  is  now 
generally  applied  to  determine  whether  or  not  the  Spirochaeta  is  in  the 
system.  The  reaction  is  due  to  certain  bodies  in  the  blood  serum  of 
syphilitic  persons  that  display  a  marked  affinity  for  lipoids  and  in  parti- 
cular, lecithin.  Many  workers  now  use,  as  antigen,  an  emulsion  of  lecithin 
or  guinea-pig  heart,  in  place  of  the  watery  emulsion  of  the  liver  obtained 
from  a  syphilitic  fetus  as  described  by  the  originators  of  the  reaction; 
the  advantages  being  that  lecithin  and  guinea-pig's  heart  are  always  on 
hand  and  alcoholic  extracts  are  more  stable  than  watery  extracts. 

The  following  is  an  outline  of  the  method  of  procedure  as  given  by 
George  Gillman  of  San  Francisco. 

i.  Antigen  (a)  (original  Wassermann);  the  liver  of  a  syphilitic  fetus  is 
cut  into  very  small  pieces  and  an  emulsion  made  of  it  by  shaking  with 
normal  salt  solution  (0.85  per  cent.)  in  the  proportion  of  one  (i)  part  of 
the  fiver  to  five  (5)  parts  of  the  salt  solution.  After  the  shaking  is  com- 
pleted, the  supernatant  liquid  is  removed  and  clarified  by  centrifugaliza- 
tion,  after  which  the  clear  liquid  is  pipetted  off,  one-half  of  i  per  cent,  of 
phenol  added  and  stored  on  ice  until  wanted  for  use. 

(b)  If  lecithin  .is  to  be  used  as  the  antigen,  it  is  prepared  as  follows: 
Make  up  a  solution  of  pure  lecithin  in  alcohol;  of  this  alcoholic  solution, 
a  quantity  equal  to  o.i  gm.  of  lecithin,  is  added  to  100  cc.  of  normal  salt 
solution.    This  is  also  stored  on  ice. 

(c)  Guinea-pig  heart  extract  is  prepared  as  follows:  The  heart  is 
rubbed  up  very  fine  in  a  mortar  (containing  ground  glass)  with  absolute 
alcohol  in  the  proportion  of  one  (i)  gram  of  the  heart  to  25  cc.  of  absolute 
alcohoL    It  is  then  heated  to  60°  C.  for  an  hour,  filtered  through  filter- 
paper  and  kept  in  the  refrigerator  ready  for  use. 

As  the  strength  of  the  antigen  will  vary  in  different  preparations,  it 
must  be  standardized  before  being  used.  It  should  be  of  such  strength 


COMMUNICABLE   DISEASES  393 

that  the  quantity  used  will  not  hemolyze  i  .o  cc.  of  a  5  per  cent,  suspension 
of  washed  lamb's  blood-corpuscles  in  the  presence  of  0.2  cc.  of  a  known 
positive  serum,  o.i  cc.  of  complement,  and  2  minimal  units  of  the  hemoly- 
tic  serum.  The  unit  is  determined  as  follows:  A  series  of  test-tubes  are 
prepared  each  containing  the  same  quantities  of  the  reagents  mentioned 
above  and  varying  amounts  of  the  antigen.  The  usual  technic  is  followed 
and  the  unit  determined  by  the  quantity  of  antigen  that  inhibited  hemoly- 
sis.  After  this  determination  the  same  antigen  must  be  tested  with  a 
known  negative  serum  used  in  place  of  the  positive  serum  and  using  double 
the  unit  of  antigen.  This  double  unit  should  not  inhibit  hemolysis  of 
the  blood  cells.  The  unit  being  determined,  the  antigen  is  so  diluted  that 
i.o  cc.  will  contain  the  unit. 

2.  Antibody. — The  blood  serum  or  cerebrospinal  fluid  of  the  syphilitic 
person.     A  sufficient  quantity  of  the  patient's  blood  is  collected  from  the 
lobe  of  the  ear  or  finger  tip,  in  any  sterile  vial  (best  in  a  Wright's  capsule) , 
aseptic    precautions,    of   course,   being   observed.     The   blood   is    then 
centrifugalized  and  the  serum  used.     The  spinal  fluid  is  obtained  in  the 
usual  manner  by  lumbar  puncture. 

3.  Complement. — The  normal  blood  serum  of  a  guinea-pig.     The  blood 
from  one  guinea-pig  is  required,  thus  making  it  necessary  to  sacrifice  one 
animal  for  each  test.     The  blood  must  be  used  fresh,  as  the  serum  loses  its 
complementing  value  if  kept  over  twenty-four  hours.     The  blood  is  defibri- 
nated,  centrifugalized,  and  the  serum  used.     If  stored,  it  should  be  frozen. 

4.  Hemolytic  Serum. — The  blood  serum  of  a  rabbit  that  has  been 
injected  with  washed  lamb's  blood-corpuscles.    The  rabbit  is  immunized 
as  follows:  The  lamb's  blood  is  first  obtained,  best  by  cutting  its  ear  and 
allowing  10  cc.  of  blood  to  run  into  30  cc.  of  a  i  per  cent,  sodium  citrate 
solution  in  normal  salt  solution.     (This  will  prevent  the  blood  from  clott- 
ing).    It  is  then  centrifugalized,  the  supernatant  fluid  pipetted  off,  and 
the  blood-corpuscles  washed  with  normal  salt  solution  by  repeated  centri- 
fugalization  and  dejection  of  the  supernatant  fluid.     Five  cc.  of  the 
washed  blood-corpuscles  are  injected  into  the  rabbit  five  (5)  or  six  (6) 
times  at  repeated  intervals  of  five  (5)  days.     On  about  the  tenth  day  after 
the  last  injection,  blood  is  taken  from  the  rabbit,  centrifugalized,  and  the 
serum  used.     Before  using  this  serum,  it  is  necessary  to  test  its  power 
after  being  inactivated  (heated  for  threequarters  of  an  hour  at  56°  C. 
to  destroy  complement).     The  test  is  made  to  determine  the  minimum 
quantity  of  the  serum  that  will  hemolyze  i  cc.  of  the  5  perfcent.  suspension 
of  lamb's  blood-corpuscles,  with  o.i  cc.  of  complement  (normal  guinea- 
pig  serum).     Various  quantities  of  the  serum  to  be  tested  are  put  in  a 
series  of  test-tubes  with  i  cc.  of  the  suspension  of  lamb's  blood  corpuscles 
and  o.i  cc.  of  the  complement  in  each  tube.     The  tubes  are  put  in  the 


394  PHARMACEUTICAL  BACTERIOLOGY 

incubator  at  37°  C.,  for  an  hour  and  then  examined  to  determine  the  smal- 
lest quantity  of  serum  that  produced  hemolysis.  (The  proper  quantity 
is  usually  i  cc.  of  a  i  in  2000  dilution,  in  normal  salt  solution.  The 
quantity  necessary  for  the  reaction  is  two  minimal  units,  thus  i  cc.  of  a 
1000  dilution  is  used  for  the  reaction).  The  dilution  used  should  never  be 
lower  than  1:1000.  If  it  happens  to  be  lower  it  will  be  necessary  to  give 
the  rabbit  a  few  more  injections  of  blood-corpuscles,  before  using  its  serum. 

5.  Lamb's  Blood  Corpuscles. — Five  cc.  of  defibrinated  lamb's  blood  are 
collected  and  washed  with  normal  salt  solution  in  the  same  way  as  the 
rabbit's  blood.  Then  a  5  per  cent,  suspension  in  normal  salt  solution  is 
made. 

The  antigen,  the  patient's  serum  and  the  hemolytic  serum  must  be  in- 
activated (to  destroy  complement)  before  using,  by  heating  them  for  three- 
quarters  of  an  hour  at  56°  C.  The  two  sera  should  be  inactivated  as  soon 
as  made. 

The  antigen,  antibody  (patient's  serum)  complement,  and  hemolytic 
serum  should  each  be  so  diluted  with  normal  salt  solution  that  i  cc.  of  the 
dilution  will  contain  the  necessary  quantities  needed  for  the  reaction. 

Technic  for  Performing  the  Reaction. — Into  a  test-tube  place  0.2  cc. 
of  the  antigen,  0.2  cc.  of  the  patient's  serum  (antibody),  and  o.i  cc. 
of  the  complement.  Incubate  at  37°  C.  for  three-quarters  of  an  hour  and 
then  add  i.o  cc.  of  the  solution  of  hemolytic  serum,  containing  two  mini- 
mal doses  and  i.o  cc.  of  the  5  per  cent,  suspension  of  lamb's  blood- 
corpuscles.  Incubate  the  whole  for  two  hours,  place  in  the  refrigerator 
over  night,  and  then  note  if  hemolysis  has  occurred.  If  the  antibody  of 
syphilis  is  present  in  the  suspected  blood  serum,  hemolysis  will  not  occur 
because  the  complement  is  " fixed"  to  the  immune  body  by  the  aid  of  the 
antigen  and  the  reaction  is  positive.  Should  the  suspected  blood  serum 
not  contain  the  specific  antibody,  hemolysis  will  occur  because  there  is  no 
immune  body  to  "fix"  the  complement,  therefore  causing  the  hemolytic 
amboceptor  (hemolytic  serum),  by  the  aid  of  the  red  corpuscles,  to  fix  the 
complement,  producing  hemolysis  and  the  reaction  is  then  negative. 

The  substances  employed  are  subject  to  many  external  influences,  and 
it  is,  therefore,  necessary  to  control  their  action.  The  controls  made  are 
necessary  in  order  to  demonstrate  that  none  of  the  employed  substances 
alone  "fix"  the  complement,  and  that  the  occurrence  of  either  a  positive  or 
a  negative  reaction,  when  testing  a  suspected  serum,  is  due  to  and  depen- 
dent upon  the  fixation  or  non-fixation  of  the  complement  by  means  of  the 
immune  body. 

The  quantity  of  antigen  used  for  the  reaction  may  have  to  be  either 
increased  or  decreased.  The  controls  will  indicate  when  a  change  is 
required  and  the  proper  quantity  necessary  is  determined  by  the  method 
given  under  the  preparation  of  the  antigen. 


COMMUNICABLE   DISEASES 


395 


There  are  many  other  tests  to  demonstrate  the  presence  in  the 
human  organism  of  syphilis;  as  the  chloride  of  gold  test  which  within 
recent  years  has  gained  much  favor  among  clinicians;  the  gum  mastic 
test  is  considered  excellent  in  the  hands  of  some  workers;  etc.  For  a 
time  laboratory  workers  recommended  a  microanalytical  method  for 
the  demonstration  of  syphilis  but  it  never  met  with  favor. 

There  are  many  other  communicable  diseases  as  measles,  mumps, 
scarlet  fever,  and  whooping  cough,  besides  the  diseases  due  to  the  attacks 
of  higher  parasites,  as  itch,  trichinosis,  tapeworm,  roundworm,  liver  flukes, 
hookworm,  etc.,  which  we  will,  however,  not  discuss  more  fully.  The 
suggestions  given  under  the  diseases  described  will  also  apply,  in  a  measure 
to  other  communicable  diseases.  Summed  up  briefly,  preventive  medicine 
direct  and  indirect,  consists  of  giving  heed  to  the  following. 

j.  Living  in  accord  with  the  most  approved  methods  of  hygiene.  This 
is  direct  preventive  medicine. 

2.  Treating  disease  in  accord  with  the  most  approved  modern  methods. 
This  is  indirect  preventive  medicine  because  it  protects  the  well  against 
infection  from  the  sick. 

The  following  table  of  communicable  diseases  giving  the  average 
period  of  incubation  (also  known  as  latent  period),  the  primary  cause, 
nature  of  communicability  and  principle  carriers  or  sources  of  infection, 
will  be  found  useful. 


Name  of  disease 

Incubation 
period, 
day 

Primary  cause 

Nature  of 
communicability 

Carrier  or  suorces 
of  infection 

Anthrax  or  wool  sorter's 
disease. 

2 

Bacillus  anthracis 

Infectious  and 
contagious. 

Cattle,  sheep. 

Bubonic  plague 

4-6 

Bacillus  pestis  .  .  . 

Infectious  

Rats,   mice  fleas, 

filth. 

Asiatic  cholera  

2-4 

Bacillus  cholerae 

Infectious       and 

Flies,     polluted 

contagious. 

water  and   food. 

Diphtheria  ... 

2—3 

Bacillus  diphtheria 

Infectious         and 

Animals,    foods, 

contagious. 

the  sick. 

Erysipelas      .  . 

4-6 

Streptococcus  py- 

Very      contagious 

Dirt,  perhaps  flies, 

ogenes. 

to  wounds. 

mosquitoes. 

Influenza,  Grippe  

1-4 

Bacillus  inflenzae. 

Infectious,        not 

Air,   and    exposed 

contagious. 

objects. 

Glanders 

3—5 

Bacillus  mallei    .  . 

Contagious       and 

Horse  and   horse- 

infectious. 

like  animals. 

Gonorrhea 

3~S 

Very     contagious. 

,  All  contaminated 

rheae. 

not    infectious. 

objects. 

Mumps  

10-16 

Unknown  

Very  infectious  .  .  . 

Air  and  the  sick. 

Malaria 

6—  10 

piasmodium   mal- 

Infectious,         not 

Mosquitos  (Anop- 

arise. 

contagious. 

heles.) 

Relapsing  fever 

5-6 

Spirochaeta    Ober- 

Infectious  

Insects,     as     bed 

mieeri. 

bugs,  etc. 

Measles 

8-9 

Very     contagious. 

Exposed  objects. 

396 


PHARMACEUTICAL  BACTERIOLOGY 


Name  of  disease 

Incubation 
period, 
day 

Primary  cause 

Nature  of 
communicability 

Carriers  or  source 
of  infection 

also  infectious. 

20—60 

Unknown  

Contagious        t  o 

wounds. 

and  other  canines 

18 

Unknown 

contagious. 

2—5 

Unknown,      p  e  r- 

Infectious       and 

haps  Protozoa. 

contagious. 

Small-pox  

12 

Unknown  

Infectious       and 

Exposed  objects. 

very   contagious. 

14—30 

Spirochaeta.'  pallida 

Very     contagious 

Exposed  objects 

especially          to 
lesions. 

A     filth     disease. 

Tetanus  lock-jaw  ........ 

2—3 

Bacillus  tetani    .  . 

Contagious  to  le- 

Dirt, infected   ob- 

sions only. 

jects  of  all  kinds. 

14. 

{Bacillus   typhosus 

Infectious       and 

Polluted         water 

contagious. 

and  food.     Flies. 

3-6 

Unknown  

Contagious  bv  in- 

Cow virus,  human 

oculation      only. 

vaccinia. 

Varicella   chicken-pox. 

14—15 

Unknown  

Contagious    

Those  affected. 

g 

Unknown  

Very  infectious 

Exposure  to  those 

affected. 

3~4 

Unknown,       plas- 

Infectious,         not 

Mosquitos  (Aedes- 

modium  ? 

contagious. 

calopus). 

Weeks, 

Bacillus  leprae.  .  .  . 

Infectious         and 

The  patients. 

months, 
years. 

contagious. 

Weeks 

Bacillus    tubercu- 

Infectious   

Sputum,          milk 

and 
longer. 

losis. 

from     tubercular 
cows. 

4 

Protozoa?  

Infectious  

Mosquito      (culex 

fatigans). 

1—2 

Micrococcus  pneu- 

Infectious   

Carried    by    per- 

monias (Diploco- 
cus. 

sons. 

g 

Amaeba         dysen- 

Infectious  

Pollusted    water 

tevise 

supply. 

6—io 

Micrococcus  meli- 

Goats'  milk,  stings 

tensis. 

of  insects. 

Beri-beri  

Months 

Micrococcus?  .... 

Infectious  

A  tropical  disease. 

Pellacrra  

? 

? 

Neither  infectious 
nor  contagious. 

The     fly     Simuli- 
um  (?) 

COMMUNICABLE  DISEASES  397 

In  some  diseases  the  mortality  rate  is  very  high,  as  in  yellow  fever, 
beri-beri,  tetanus,  cholera,  plague  and  leprosy.  In  others  it  is  low,  as  in 
syphilis,  gonorrhea,  malaria,  whooping  cough,  mumps  and  varicella.  In 
certain  diseases  the  prognosis  is  rather  uncertain,  the  mortality  rate  being 
high  at  times  and  again  low,  as  in  scarlatina,  small-pox,  measles  and  grippe. 
In  the  recent  grippe  or  influenza  pandemic  the  mortality  rate  was  unus- 
ually high.  In  this  disease  the  mortality  rate  is  high  at  the  early  periods 
of  the  invasion,  gradually  growing  less  and  less  severe.  It  may  be  that 
the  causative  organism,  whatever  it  may  be,  loses  in  virulency  in  its 
passage  through  the  hosts.  The  hemorrhagic  form  of  smallpox  has  a 
mortality  rate  of  nearly  100  per  cent,  whereas  some  epidemic  forms  of 
this  disease  are  very  mild,  showing  practically  100  per  cent,  recoveries. 
The  so  called  black  scarlatina  shows  a  very  high  mortality  rate.  Some 
epidemics  of  measles  show  a  comparatively  high  mortality  rate.  Some 
diseases  run  a  somewhat  variably  rapid  course  as  pneumonia,  diphtheria, 
spinal  meningitis,  bubonic  plague  and  Asiatic  cholera,  ending  either 
in  death  or  recovery.  Other  diseases,  as  scarlet  fever,  measles  and 
diphtheria  may  have  after-effects  or  sequelae  which  often  assume  a  chronic 
course  and  may  finally  result  in  death.  Certain  diseases  run  a  regular 
course  which  varies  but  little  as  to  the  sequence  of  symptoms  and  duration, 
as  typhoid  fever  (five  weeks).  Others  run  a  variably  chronic  course, 
ending  either  in  death  or  recovery,  as  pellagra  and  malaria.  Some  diseases 
are  very  persistent,  difficult  to  eradicate  from  the  system,  showing  certain 
effects  even  to  the  third  and  fourth  generations,  as  tuberculosis  and  syphilis 
Malaria  leaves  certain  after-effects,  as  enlarged  spleen  ("ague  cake")> 
which  may  persist  through  life. 

Savage  races  are  peculiarly  susceptible  to  certain  diseases,  as  tubercu- 
losis, small-pox,  gonorrhea  and  syphilis  and  peculiarly  enough  these 
diseases  did  not  originate  with  primitive  peoples,  but  with  advanced 
civilization,  though  of  great  antiquity. 

DISSEMINATION  OF  DISEASE 

The  manner  in  which  disease  is  spread  has  been  indicated  in  the  pre- 
cedeing  but  the  subject  has  not  been  fully  discussed.  The  student  of 
pharmacy  should  have  a  course  in  general  sanitary  science  and  in  pre- 
ventive medicine.  The  general  principles  of  immunity  have  been  dis- 
cussed and  the  more  important  communicable  diseases  have  been  briefly 
outlined,  but  the  vast  subject  of  preventive  medicine  has  not  been  touched 
upon.  This  must  be  taken  up  in  a  separate  course. 

The  agents  and  agencies  concerned  in  the  spreading  of  communicable 
diseases  and  others,  constitute  the  groundwork  of  preventive  medicine. 


398 


PHARMACEUTICAL  BACTERIOLOGY 


COMMUNICABLE   DISEASES  399 

Of  almost  equal  importance  is  a  knowledge  of  the  agencies  which  man 
may  make  use  of  in  the  control  of  the  agencies  of  disease  dissemination. 
The  proper  dissemination  of  a  knowledge  of  sanitary  science  is  of  even 
greater  importance  than  is  sanitary  legislation. 

The  table  on  opposite  page  and  the  classification  of  disease  carriers 
and  the  outline  of  a  course  in  sanitary  science  will  serve  to  indicate 
the  ground  that  is  to  be  covered  in  a  course  for  students  of  pharmacy. 


CHAPTER  XVIII 

SUGGESTIONS  ON  A  MICROANALYTICAL  AND  BACTERIOLO- 
GICAL LABORATORY  FOR  THE  PHARMACIST 

i.  The  Qualifications  of  the  Analyst 

What  type  and  grade  of  scientific  or  analytical  work  should  the 
properly  trained  pharmacist  be  prepared  to  do?  What  should  his  special 
training  and  qualifications  be  in  order  that  he  may  do  special  work? 
These  questions  have  been  discussed  within  recent  years  by  the  leading 
pharmacists  in  the  United  States.  It  is  not  within  the  province  of  a  work 
of  this  kind  to  discuss  pharmaceutical  education,  nevertheless  the  following 
suggestions  are  in  place. 

1.  The  pharmacist  who  has  had  a  thorough  training  in  an  adequately 
equipped  college,  assuming  that  he  has  mental  aptitude,  should  be  pre- 
pared to  do  special  work  along  the  following  lines. 

(a)  Chemistry  and  toxicology. 

(6)  Pharmacy  and  pharmaceutical  manufacture. 

(c~)  Pharmacognosy  and  microanalysis. 

(d)  Bacteriology. 

2.  For  some  time  to  come  the  specialists  in  pharmacy  will  tend  to 
work  in  more  than  one  branch;  or  rather,  the  special  endeavor  will  be 
developed  through  correlated  branches  of  science.     Thus  the  toxicologist 
will  advance  through  chemistry  and  pharmacology.     The  pharmacogno- 
sist  through  botany  and  materia  medica.     The  bacteriologist  through 
sanitary  science  and  microanalysis. 

3.  The  present  arrangement  of  courses  in  our  leading  colleges  of 
pharmacy  is  such  that  the  following  specialists  will  arise  from  the  ranks 
of  the  limited  number  of  promising  students. 

(a).  Toxicologists,  rather  than  chemists  or  pharmacologists. 

(b) .  Pharmacognosists,  rather  than  botanists  or  specialists  in  materia 
medica. 

(c.)  Microanalysts,  rather  than  bacteriologists  or  sanitary  officers. 

It  is  true  that  a  number  of  specialists  employed  in  state  and  munici- 
pal laboratories  and  in  pharmaceutical  laboratories  (pharmaceutical 
manufacture)  have  arisen  from  the  ranks  of  the  students  of  pharmacy,  and 
pharmaceutical  chemistry,  but  it  is  nevertheless  a  fact  that  the  college 
curriculum  is  such  as  to  train  and  qualify  especially  along  the  lines  in- 
dicated. Our  authoritative  pharmacologists  are  without  exception  from 

400 


MICRO  ANALYTICAL  AND  BACTERIOLOGICAL   LABORATORY        401 

the  ranks  of  those  holding  higher  university  degrees.  Our  eminent  bacteri- 
ologists and  sanitarians  are  largely  men  with  medical  and  university 
degrees.  Our  leading  botanists  are  from  universities.  The  college  of 
pharmacy  alone  (but  not  by  any  means  all  of  them)  specialize  along  the 
lines  of  pharmacy,  pharmacognosy,  toxicology  and  microanalysis.  To  a 
lesser  degree  also  in  materia  medica  and  in  the  chemical  testing  and  assay- 
ing of  medicamenta.  A  few  of  the  colleges  prepare  fairly  well  qualified 
sanitary  assistants,  if  not  sanitary  directors.  Other  branches  of  science 
which  may  be  taught  in  a  college  of  pharmacy,  such  as  botany,  physiology, 
pharmacology,  sanitary  science,  and  bacteriology,  are  simplified  and 
specially  modified  presentations  of  like  course  as  given  in  medical  schools 
and  in  universities.  The  common  every  day  practices  of  pharmacy, 
such  as  filling  prescriptions,  pill  rolling  and  tablet  making,  cannot  be 
rated  as  science.  These  operations  come  under  the  head  of  art. 

4.  As  to  microanalysis  and  bacteriology  considered  as  practical 
working  specialties,  the  following  is  offered. 

(a).  The  two  specialties  go  together  and  merge  into  one  another.  The 
microanalyst  should  be  qualified  to  do  bacteriological  work,  and  vice 
versa,  the  bacteriologist  must  be  prepared  to  do  microscopical  work. 

(b).  The  college  course  in  microanalysis  must  be  comprehensive  and 
should  serve  as  a  basis  for  the  bacteriological  work  (the  direct  bacteriolo- 
gical methods). 

(c) .  The  course  in  bacteriology  should  be  well  flanked  by  the  correlated 
sciences,  zymology,  parasitology,  serology,  immunology  and  general 
sanitary  science. 

The  following  outline  will  serve  to  explain  the  scope  of  the  correlated 
sciences,  microanalysis,  bacteriology  and  sanitary  science,  and  the  prepa- 
ration which  is  necessary  to  do  efficient  laboratory  work.  The  subjects 
outlined  are  to  be  taught  in  the  third  and  fourth  years  of  a  college  of 
pharmacy  having  the  usual  university  entrance  requirements. 

Course  I.  General  Microanalysis. — Four  hours  of  laboratory  work 
each  week,  during  the  entire  college  year.  This  course  is  to  be  given 
during  the  third  year  of  the  full  four  year  course  and  is  to  follow  the 
laboratory  course  in  pharmacognosy  and  in  plant  histology. 

A.  THE  MICROSCOPICAL  EXAMINATION  OF  FIBER,  FOODS  AND  DRUGS 

I.  Examination  of  Fiber, 
i.  Vegetable  Fiber. 

(a)  Cotton.     Cotton  cloth.     Mercerized  cotton  cloth 
(6)  Paste  board.     Wrapping  paper.     Tissue  paper. 

(c)  News-paper.     Filter  paper.     Etc. 

(d)  Book  paper.     Bank  note.     Etc. 

(e)  Writing  paper. 

26 


402  PHARMACEUTICAL  BACTERIOLOGY 

(/)    Cordage.     Thread.     Etc. 
(g)  Hemp  fiber  and  cloth. 
(A)  Linen  cloth.     (Linen  tester.) 
(i)   Artificial  (viscose)  silk. 

2.  Animal  Fiber, 
(a)  Human  hair. 

(&)  Hair  of  other  animals.     Wool.     Bristle. 

(c)  Woolen  cloth. 

(d)  Gamers  hair.     Alapaca.     Mohair. 

(e)  Silk  fiber.     Silk  cloth. 
(/)  Artificial  (gelatine)  silk. 

3.  Mixed  Fiber.     Animal  and  Vegetable. 
(a)  Government  note.     Bank  note. 
(&)  Felt  paper. 

(c)  Shoddy. 

(d)  Mixed  cloth  (wool  and  cotton). 

4.  Inorganic  Fiber. 

(a)  Glass  fiber.     Glass  wool. 
(&)  Asbestos. 
(c)   Metal  fiber. 

II.  Commercial  Starches. 

1.  Corn  starch.  Rice  starch.  Wheat  starch. 

2.  Potato  starch.  Sweet  potato  starch.  Banana  starch. 

3.  Arrowroot  starches,  etc. 

III.  Dextrins.     A  comparative  study  should  be  made  of  the  different  kinds  of  dextrins 
in  order  to  determine  the  source  of  the  starch  used,  the  degree  and  character  of  the 
dextrinization,  etc. 

IV.  Starch  Fillers.     A  study  of  starch  fillers  used  in  sausage  meats. 

V.  Ice    Cream    Fillers.     Kinds.     Starch    fillers.     Gum    arabic.     Tragacanth,    etc. 

Fillers  combined  with  milk  coagulants  (rennet). 

VI.  Flours  and  Meals. 

1.  Cereal  flours.     A  critical  comparative  study  should  be  made  of  wheat,  rye,  rice 
and  barley  grains  and  the  flours  made  therefrom.     The  hand  gluten  test.    The 
Bamihl  test  and  the  Winton  modification  of  the  Bamihl  test.     Processed  flours 
and  chemical  tests  for  bleached  flours.    Polished  rice. 

2.  Oat  meal  and  corn  meal.     Make  a  comparative   study.     Note   the   distinct 
polarizing  bands  in  the  corn  starch. 

3.  Buckwheat  flour.     Italian  Buckeye  meal,  etc. 

4.  Pancake  flours.     Mixed  flours.     Make  estimates  of  the  percentages  of  the  dif- 
ferent flours  in  the  compound  or  mixture. 

5.  Banana  meal.    Squash  meal,  etc. 

VII.  Comparative  Study  of  Brans. — Wheat,  rye,  rice,  barley  and  corn  brans.  Middlings. 

VIII.  Cotton  Seed  Cake    Linseed  Cake.     A  comparative  study. 

IX.  Prepared  Starches,  Flours,  and  Meals. 

1.  Spaghetti,  macaroni,  noodles. 

2.  Sago,  etc. 

X.  Bread  and  Pastry.     Examine  as  to  the  identity  of  the  materials  used,  as  to  kind 

of  flour,  etc. 

1.  Breads,  biscuits,  rolls,  etc.     Examine  as  to  the  identity  of  starch,  use  of  mixed 
flours,  absence  or  presence  of  yeast  cells,  etc. 

2.  Cake,  cookies,  "Arrowroot  biscuits,"  etc. 


MICROANALYTICAL  AND  BACTERIOLOGICAL   LABORATORY         403 

XI.  Breakfast  Foods.    These  are  to  be  examined  as  to  the  material  used,  ascertaining 
manner  of  manufacture  and  the  absence  or  presence  of  substances  declared  on  the 
label. 

1.  Flaked  corn  and  wheat. 

2.  Rolled  corn  and  wheat.     Mixtures. 

3.  Puffed  rice  and  wheat.     Manner  of  manufacture.     Chinese  and  Hindoo  purled 
rice. 

4.  "Cream  of  wheat,"  "Carnation  mush,"  etc. 

5.  Shredded  wheat,  grape  nuts,  etc. 

XII.  Baby  and  Invalid  Foods.     These  are  to  be  examined  as  to  the  presence  of  flours, 
of  unaltered  starch,  as  to  the  identity  of  starch,  use  of  milk  or  cane  sugar,  presence 
of  dried  milk,  of  casein,  etc. 

1.  Dried  milk  and  casein.     Pure  and  mixed. 

2.  Starchy  baby  foods. 

3.  Horlick's  malted  milk. 

4.  Borden's  condensed  and  malted  milk. 

5.  Eskay's  food. 

6.  Peptogenic  milk  powder,  etc. 

XIII.  Spices  and  Condiments.     These  are  to  be  examined  as  to  identity  and  quality 
(organoleptic  testing)  and  as  to  absence  or  presence  of  adulterants. 

1.  Pepper. — Black  and  white.     Processed  white  pepper  (bleached). 

2.  Capsicums. — Hungarian,  Mexican,  American,  etc. 

3.  Allspice  and  allspice  stems. 

4.  Cloves  and  clove  stems.     Exhausted  cloves. 

5.  Nutmeg,  mace.     False  mace.     Adulterants. 

6.  Cinnamons.     Adulterants  and  quality. 

7.  Mustard.    Prepared  mustards.     Mustard  hulls. 

8.  Herbaceous  condiments.     Marjoram,  sage,  thyme,  etc. 

9.  Umbelliferous  spices.     Curry  powders,  etc. 

XIV.  Dairying  Products. 

1.  Milk.     Make  a  critical  comparative  study  of  normal  cow's  milk,  pasteurized 
milk,  boilt  miik,  evaporated  milk  and  condensed  milk,  including  bacterial  con- 
tent, presence  of  blood  corpuscles,  pus  corpuscles,  sediment,  etc. 

2.  Sour  milk,   klabbered  milk,   buttermilk.     Examine  as  to  bacteria  and  other 
organisms  present.     Dutch  hydrogen  peroxide  test.     Examination  of  washed  and 
centrifugalized  samples. 

3.  Cream.     Whole  milk.     Half  milk.     Skimmed  milk. 

4.  Butter  and  butter  substitutes. 

5.  Cheese  and  cheese  parasites. 

XV.  Home  Drinks. 

1.  Coffee.     The  normal  and  roasted  bean.     Ground  coffee.     Coffee  substitutes. 
Dekofa.     Cereal  coffee.     A  careful  study  of  ground  coffees  and  their  more  com- 
mon   admixtures    and    adulterants.      A    study    of    coffee    substitutes    as    to 
composition. 

2.  Teas.     Qualities  and  grades.     Government  standards  and  tests.     Tea  culture 
in  the  United  States. 

(a)  Coloring  substances.     Reed  and  West  tests  for  color. 

(b)  Exhausted  teas.     Tea  adulterants.     Japanese  teas. 

(c)  Tea  substitutes. 

3.  Cocoas  and  Chocolates. 


404  PHARMACEUTICAL  BACTERIOLOGY 

(a)  Cocoas  and  chocolates.     Method  of  manufacture. 

(6)  Cocoa  shells. 

(c)  "Soluble  cocoas." 

(d)  Cocoa  butter. 

(e)  Adulterants  of  cocoa  and  chocolates. 

XVI.  Food  Products,  Animal  and  Vegetable.     Samples  may  be  secured  from  private 
homes,  grocers  and  canneries.     They  are  to  be  examined  as  to  identity,  quality 
and  purity  and  the  findings  recorded  on  special  report  cards.     In  the  examination 
of  these  substances  the  polarizes,  the  micrometer  scale,  the  Thoma-Zeiss  hemacy- 
tometer  (Turck  ruling)  and    other  necessary  apparatus  are  used.     The  Lager- 
heim  sublimation  tests  for  benzoic  acid  and  salicylic  acid  and  the  Curcuma  thread 
test  for  boric  acid  and  the  starch  paper  test  for  sulphurous  acid  are  used. 

1.  Canned  meats.     Canned  fish.     Anchovy  pastes,  etc.     Examine  for  mold  and 
bacterial  contamination  and  the  presence  of  preservatives. 

2.  Sausage  meats.     Examine  for  starches  and  starch  fillers,  preservatives  and  added 
coloring  substances. 

3.  Jams  and  jellies.     Examine  as  to  identity,  use  of  green  fruit,  fruit  refuse,  preser- 
vatives, yeast,  bacteria,  mold.     Presence  of  agar  or  other  fillers. 

4.  Catsups  and  tomato  pastes.     Examine  for  preservatives,  mold,  bacteria  and 
yeast  cells,  tomato  refuse,  starch,  etc. 

5.  Preserved  and  pickled  fruits.     Examine  for  bacteria  and  yeast,  for  sulphurous 
acid  (bleached  fruits)  and  for  preservatives.     (Leaks  and  swells). 

XVII.  Candies.     Qualities    and    Grades.     They   are   to   be   examined   for    various 
fillers  (starch,  flour,  gums,  etc.),  nature  of  coating,  coloring  matter,  impurities,  etc. 

XVIII.  Vegetable  Drugs,  Crude  and  Powdered.     Compound  Powders,  Pills,  Tablets, 
Extracts.     Examine  as  to  quality  and  purity,  ash  content  (including  test  for  acid 
insoluble  ash),  fineness  of  powders,  organoleptics  tests,  etc. 

1.  Powdered  vegetable  drugs. 

2.  Compound  powders.     Dusting  powders.     Face  powders. 

3.  Cattle  powders. 

4.  Poultry  powders. 

5.  Extracts,  solid  and  fluid. 

6.  Medicinal  teas. 

7.  Pills. 

8.  Tablets. 

9.  Crude  drugs.     Pressed  herbs. 

10.  Patent  and  proprietary  preparations  of  an  organic  nature. 

11.  Calomel,  charcoal,  mercury,  sulphur. 

12.  Pastes,  plasters,  ointments. 

13.  Snuffs,  cigarettes,  tobaccos. 

14.  Unknowns. 

The  sequence  of  the  several  operations  of  the  complete  analysis  of  a  sample  of  pow- 
dered vegetable  drug  may  be  given  as  follows: 

1.  Noting  the  condition  of  the  seals  of  the  sample  or  package.     Breaking  the  seals. 

2.  Thoroughly  mixing  the  sample.     Selecting  an  average  sample. 

3.  Organoleptic  testing  (consistency  or  feel,  color,  odor,  taste). 

4.  Determining  the  fineness  by  means  of  a  suitable  nest  of  sieves. 

5.  Preliminary  examination  of  the  average  sample  and  of  the  samples  upon  the  dif- 
ferent sieves,  using  pocket  lens,  tweezers,  etc.     Organoleptic  testing  of  individual 
fragments,  etc. 


MICROANALYTICAL  AND  BACTERIOLOGICAL   LABORATORY        405 

6.  Special  examination  (macroscopical  and  microscopical)  of  the  several  portions  on 
the  different  sieves  if  thought  desirable  or  necessary. 

7.  Again  mixing  the  several  portions  on  the  several  sieves  and  reducing  to  uniform 
fineness,  if  thought  desirable  or  necessary. 

8.  Complete  and  thorough  microscopical  examination. 

9.  Ash  determination  if  thought  desirable. 

10.  Acid  insoluble  ash  determination  if  thought  desirable, 
n.  Special  tests  if  thought  desirable. 

12.  Recording  the  results  of  the  analysis. 


B.  THE  MICROSCOPICAL  EXAMINATION  OF  THE  BODY 

I.  External  Tegument. 

1.  Hair  and  scalp. 

(a)  Macroscopic  parasites.     Eggs,  larvae. 
(6)  Microscopic  parasites. 

(c)  Sebaceous  deposits  and  dirt. 

(d)  Epithelial  cells  and  other  tissue  cells. 

(e)  Evidences  of  diseased  tissues. 
(/)    Powders  used,  etc. 

(g)  Hair  tonics,  ointments,  hair  oils,  etc. 

2.  Face. 

(a)  Eye  secretions,  normal  and  abnormal. 
(6)  Secretions  of  the  ear. 

(c)  Facial  hair. 

(d)  Face  lotions,  ointments,  etc. 

(e)  Face  powders,  etc. 

(/)    Eruptions  and  other  abnormal  conditions. 

3.  Skin — Normal  and  abnormal.     Proceed  much  as  for  Hair  and  Scalp. 

4.  Finger  Nail  Deposits. 
(a)  Nature  of  deposits. 

(&)  Interpretations  of  deposits. 

II.  Internal  Tissue. — Normal   and  Abnormal. 

1.  Muscular  Tissue. 

(a)  Normal. 

(b)  Parasite— Trichinae,  etc. 

(c)  Pathological  conditions. 

2.  Nervous  Tissue. 

(a)  Spinal  cord — Negri  bodies  in  rabies. 
(&)  Brain. 

(c)  Fibers  and  ganglia. 

(d)  Terminal  nerve  elements. 

3.  Osseous  Tissue. 

(a)  Osseous  elements. 
(&)  Periosteum. 
(c)   Marrow. 

4.  Cartilaginous  Tissue.     Kinds. 

5.  Connective  Tissue. 

6.  Adipose  Tissue. 


406  PHARMACEUTICAL  BACTERIOLOGY 

III.  Digestive  Tract. 

1.  Mouth  and  Teeth. 

(a)  Epithelium  and  other  normal  tissue  elements. 

(b)  Food  particles. 

(c)  Bacterial  flora. 

(d)  Pathological  conditions. 

2.  Stomach. 

(a)  Normal  contents,  digestion,  action  of  ferments. 

(b)  Vomited  material. 

(c)  Pathological  conditions. 

3.  Intestines. 

(a)  Small  intestines. 

(b)  Large  intestines.     Colon.     Faeces. 

(c)  Parasites. 

(d}  Pathological  conditions. 

(e)  Test  diets,  Value  and  significance  of. 

IV.  Genito-urinary  Tract.     Male  and  Female.     Normal  and  Abnormal. 

1.  Epithelial  cells,  etc. 

2.  Urinary  sediments. 

3.  Pathological  secretions. 

V.  Blood  Work. 

1.  Normal. 

2.  Pathological 

3.  Blood  counting. 

VI.  Respiratory  Tract. 

1.  Nasal  secretions. 

2.  Expectorations  and  Secretions. 

(a)  Buccal  and  pharyngeal.     Throat. 
(6)  Bronchial  sputum. 
(c)  Pulmonary  sputum. 

"  C.  URINARY  SEDIMENT.     MICROSCOPICAL  EXAMINATION 

This  outline  includes  both  the  organized  and  the  unorganized  sediments  and  deposits. 
I.  Crystalline  and  Amorphous  Chemical  Deposits  and  Sediment. 

1.  Uric  acid,  crystalline. 

2.  Uric  acid  compounds. 

(a)  Acid  sodium  urate,  (generally  amorphous,  occasionally  crystalline). 
(6)  Acid  potassium  urate  (amorphous). 
(c)  Acid  calcium  urate  (amorphous). 
(d}  Acid  ammonium  urate  (crystalline). 

3.  Calcium  oxalate  (crystalline). 

4.  Earthy  phosphates. 

(a)  Ammonium-magnesium  phoshate  (crystalline). 

(b)  Calcium  phosphate  (amorphous  and  crystalline). 

5.  Calcium  carbonate  (crystalline). 

6.  Calcium  sulphate  (crystalline). 

7.  Leucin,  tyrosin,  cystin  (crystalline). 

8.  Cholesterin  (crystalline). 


MICROANALYTICAL   AND   BACTERIOLOGICAL    LABORATORY         407 

II.  Accidental  Urinary  Inclusions. 

1.  Starch  granules. 

2.  Vegetable  cells  and  tissues.     Faecal  matter,  etc. 
(a)  Ducts  and  vessels. 

(6)  Sclerenchyma  cells. 

(c~)  Parenchymatous  tissue. 

(d)  Bast  fibers. 

(e)  Cork  cells. 
(/)  Cotton  fibers. 
(g)  Lycopodium. 
(h)  Linen  fiber. 

(»)    Seeds  and  seed  tissue. 

3.  Animal  fiber  and  elements. 
(a)  Hair  and  wool. 

k(b)  Fragments  of  feathers, 
(c)   Scales  of  moths. 
(d)  Cartilage  cells. 
(e)   Fat  globules. 
(0  Muscle  fiber, 
(g)  Fibrous  tissue. 

III.  Urinary  Concretions. 

1.  Uric  acid  calculi. 

2.  Calcium  oxalate  calculi. 

3.  Mixed  calculi. 

4.  Platinum  foil  tests. 

(a)  Ignition. 

(b)  Behavior  with  HC1. 

IV.  Casts. 

1.  Hyaline. 

(a)  Pure  hyaline. 
(6)  Fibrinous. 
(<?)  Waxy. 

2.  Granular. 

(a)  Fine. 

(b)  Coarse. 

(c)  Pigmented. 

3.  Epithelial. 

4.  Fatty. 

5.  Blood. 

6.  Pus. 

7.  Bacterial. 

8.  Mixed. 

9.  Crystalline  (organic  base). 
(a)  Urates. 

(6)  Oxalates. 
(c)   Cystin. 

False  casts  or  cylindroids. 
Mucus  Threads. 
Prostatic  Plugs. 


408  PHARMACEUTICAL  BACTERIOLOGY 

VIII.  Blood.     Smoky  Urine.     Hematuria. 

1.  Normal  blood  corpuscles. 

2.  Crenated  corpuscles. 

3.  Phantom  corpuscles. 

4.  Corpuscles  associated  with  casts. 

5.  Hemin  crystals. 

6.  Blood  clots. 

IX.  Epithelial  Cells.    Normal  and  Abnormal. 

1.  In  order  of  size. 

(a)  Vaginal  (flat  cuboidal  and  columnar.     Usually  associated  with  bacteria). 
(6)  Bladder. 

(c)  Cervix. 

(d)  Urethra. 

(e)  Renal  pelvis. 
(/)   Ureters. 

(g)  Prostate. 

(A)  Kidney  tubules. 

2.  As  to  form. 

(a)  Flat  or  squamous. 

(£)  Cuboidal  (with  or  without  fat  granules  and  analogous  to  put  corpuscles). 

(c)  Columnar  or  cylindric. 

(d)  Irregular. 

3.  As  to  quantity  or  numbers. 
(a)  Normal. 

(6)  Excess  of  squamous  (as  in  irritation  and  inflammation), 
(c)  Excess  of  cuboidal  (as  in  chronic  inflammation.     Ulceration,  with  pus  and 
blood  corpuscles). 

X.  Pus  Corpuscles— Glycogenic  Reaction. 

1.  Normal. 

2.  Amoeboid. 

3.  Inclusions  of  the  cells. 

4.  Decomposition  changes,  etc. 

XI.  Mucus  Corpuscles. 

XII.  Amyloid  Bodies. 

XIII.  Spermatozoa. 

XIV.  Micro-Organisms.     Normal  and  Pathological. 

1.  Urethral  and  vaginal  bacteria — Normal. 

2.  Air  infection  of  the  urine. 

3.  Pathological  infection  of  the  urinary  tract. 

4.  Micrococcus  urese. 

5.  Yeasts. 

6.  Molds. 

7.  Pathogenic  bacteria. 

(a)  Streptococcus  (Staphylococcus)  pyogenes. 

variety  albus. 
variety  citreus. 
variety  aureus. 

(b)  Typhoid  bacillus. 

(c)  Anthrax  bacillus. 

(d)  Erysipelas. 

(e)  Tuberculosis. 


MICRO  ANALYTICAL  AND  BACTERIOLOGICAL  LABORATORY        409 

(/)    Colon  bacillus. 
(g)  Gonococcus. 

XV.  Animal  Parasites.     Rare. 

1.  Mites. 

2.  Echinococcus. 

3.  Filaria  sanguinis  hominis. 

4.  Distoma  haematobium. 

5.  Ascarides. 

XVI.  Tumor  elements. 

XVII.  Toxic  Substances.     Urotoxic  Coefficient. 

1.  Toxins. 

2.  Ptomaines. 

3.  Leucomaines. 

D.  EXAMINATION  OF  GONORRHEAL  DISCHARGES  FOR  THE 
GONOCOCCUS 

I.  Securing  the  Sample. 

II.  Mounting  and  Staining  the  Sample. 

III.  Examining  the  Slide  Mount. 

1.  Intercellular  diplococci. 

2.  Intracellular  diplococci. 

E.  EXAMINATION  OF  TUBERCULAR  SPUTUM 

I.  Securing  the  Sample. 

II.  Mounting  and  Staining  the  Sample. 

III.  Examining  the  Slide. 

1.  Tubercle  bacilli. 

2.  Associated  organisms — Mixed  infections. 

F.    EXAMINATION  OF  FAECES 

I.  Preparing  the  Sample. 

1.  Diluting  and  washing. 

2.  Centrifugalizing. 

II.  Normal  Faeces. 

1.  Undigested  Food  Particles. 

(a)  Vegetable  tissues. 

(b)  Fruit  seeds. 

(c)  Fat  cells. 

(d)  Muscular  tissue. 

(e)  Starch,  etc. 

2.  Bacteria. 

3.  Epithelial  cells. 

III.  Abnormal  Faeces. 

1.  Dysentery. 

2.  Typhoid  fever. 

3.  Ulcerations  and  inflammations.     Blood  and  pus  corpuscles,  etc. 

4.  Intestinal  parasites. 


410  PHARMACEUTICAL  BACTERIOLOGY 

G.  BLOOD  COUNTING 

I.  The  Thoma-Zeiss  Hemacytometer.     How  Used.     Making  the  Dilutions. 

II.  Securing  the  Blood  Samples. 

III.  Mounting  the  Sample. 

IV.  Counting  the  Corpuscles. 

1.  The  red  corpuscles. 

2.  The  white  corpuscles. 

V.  Examining  the  Fresh  Blood. 

1.  Manson  Method. 

2.  Ross  method. 

VI.  Preparing  and  Staining  dried  Blood  Films. 

VII.  Blood  Glycogen  Test.     lodophilia. 

VIII.  Pathogenic  Organisms  in  the  Blood. 

The  following  blank  report  sheet  should  be  used.  The  sample  reports 
given  will  indicate  how  these  are  to  be  filled  out,  based  upon  the  results 
of  the  analysis: 

Form  No.  i.  Blank  report  sheet  for  the  microscopical  examination  of  organic  drugs 
and  dry  food  substances. 

No.     (Laboratory  or  other  serial  number). 
Label  : 

Sample  received Sample  examined 

Conditions  of  wrappings  and  seals 

Organoleptic  tests 

Consistency  of  Feel 

Color 

Odor 

Taste 

Adjunct  Tests 

Ash % 

Acid-insoluble  ash % 

Sand  (beaker  test) % 

Special  Tests 


Microscopical  Findings. . 


Conclusions . 


,  Analyst. 


MICROANALYTICAL   AND   BACTERIOLOGICAL    LABORATORY        411 

The  following  is  a  sample  report  analysis  using  the  proposed  report 
card: 

No.:     5432- 

Label:    Broken  Senna,  U.  S.  P.,  John  Smith  &•  Co.,  Kalamazoo,  Michigan. 
Sample  received:     August  15,  1912.     Sample  examined:     August  20,  1912. 
Conditions  of  wrappings  and  seals:     Good. 

Organoleptic  Tests 

Consistency  or  Feel:     Dry,  gritty,  sandy,  dirty. 
Color:     Not  unusual. 
Odor:     Senna-like. 
Taste:     Sandy,  gritty. 

Adjunct  Tests 

Ash:     19.6%. 
Acid-insoluble:     9.4%. 

Sand  (beaker  test):     9%,  sand  and  small  pebbles. 
Special  Tests:    Pebbles  picked  out  by  hand.     About  4%  senna  seeds  and  pod  fragments 

and  stems  present. 

Microscopical  Findings:  The  histological  characters  of  African  senna.  Stem  tissue 
excessive.  Sand  and  dirt  excessive.  Senna  seeds  and  pods  present  in  considerable 
quantity. 

Conclusions:  Adulterated  with  sand,  pebbles,  senna  seeds,  senna  pods  and  stems  25%. 
Misbranded  becauses  labeled  U.  S.  P.,  whereas  it  is  below  the  U  S.  P.  standard. 

RICHARD  ROE,  Analyst. 

Form  No.  II.     Blank  report  sheet  for  the  microscopical  examination  of  catsups, 
jams,  jellies,  etc. : 

(No.,  label,  dates,  condition  of  seal  and  organoleptic  tests,  as  for  Form  No.  i.) 
Adjunct  Tests. 

Sublimation  tests  for 

Benzoic  acid 

Salicylic  acid 

Boric  acid  (curcuma  thread) 

Iodine  reaction 

Intracellular 

Extracellular 

Special  Tests 

Microscopical  Findings. 
General. . 


Cytometric  counts. 

Dead  yeast  cells per  cc. 

Living  yeast  cells per  cc. 

Bacteria  * . .  .  .' per  cc. 

Mold  (hyphal  fragments  and  clusters) per  cc. 

Mold  spores per  cc. 

Living  yeasts per  cc. 

JThe  total  count,  inclusive  of  rod  shaped  forms,   coccus  forms,  etc.,  should  be 
given. 


412  PHARMACEUTICAL  BACTERIOLOGY 

Bacteria per  cc. 

Mold  (hyphal  fragments) per  cc. 

Mold  spores per  cc. 

Smut  spores per  cc. 

Conclusions . . 


Analyst. 

We  may  give  an  example  of  a  report  as  follows: 

FORM  No.  II 
Lab.  No.  462. 

Label:    Pure  currant  jetty.     Made  by  Smith,  Jones  &  Co.,  Nantucket,  Wis. 
Sample  received  August  5,  1914.     Sample  examined  August  5,  1914. 
Condition  of  seals:    Good,  unbroken  sample. 
Organoleptic  tests:    Not  conclusive. 

Consistency  or  feel:    Poorly  jellied. 
Color :     Normal  for  Currant  jelly. 
Odor:    Faint,  somewhat  disagreeable. 
Taste :     Not  characteristic,  bitterish,  quite  acid. 
Adjunct  tests. 

Sublimation  tests  for 

Benzoic  acid:    Negative. 
Salicylic  acid :     Very  marked. 
Boric  acid  (curcuma  thread) :    Negative. 
Iodine  reaction :     Very  marked. 
Intracellular :    Negative. 
Extracellular:    Positive,  very  marked. 

Special  tests :    Salicylic  acid  color  reaction,  with  ferric  chloride  very  marked. 
Microscopical  examination. 

General.     Some  apple  tissue  (window  cells  and  pulp  cells)  and  currant  tissue 

sclerenchyma  present.     Added  wheat  starch  about  5  per  cent. 
Cytometric  counts. 

Dead  yeast  cells,  80,000,000 per  cc. 

Living  yeast  cells,  none per  cc. 

Bacteria,  600,000,000 per  cc. 

Mold  (hyphal  fragments  and  clusters),  84,000 per  cc. 

Mold  spores,  5,000,000 per  cc. 

Smut  spores,  none per  cc. 

Conclusions:  Misbranded.  Adulterated  with  apple  and  with  wheat  starch  and  made 
from  fermented  and  decomposed  material,  preserved  with  salicylic  acid.  Not  fit  for 
human  consumption  because  of  the  quantity  of  yeast,  mold  and  bacteria  present. 

JOHN  DOE,  Analyst. 

Course  II.  Bacteriological — Quantitative  and  Qualitative  Determi- 
nations of  Organisms  in  Foods  and  Drugs. 

A  |  course  in  general  laboratory  technic  is  the  necessary  preparation 
to  this  course.  Such  preparatory  course  should  be  given  during  the 
third  year  of  the  full  four  year  course,  which  would  mean  that  the 
present  course  must  be  given  during  the  fourth  year. 


MICRO  ANALYTICAL  AND  BACTERIOLOGICAL  LABORATORY        413 

A  laboratory  course  of  at  least  one  hour  each  day  extending  through- 
out the  entire  college  year.  The  time  necessary  to  do  the  laboratory 
work  will  vary  from  day  to  day.  The  work  is  to  be  supplemented  by 
lectures,  special  readings  and  seminar  work.  The  laboratory  methods 
employed  are  those  of  the  Laboratory  Section  of  the  American  Public 
Health  Association,  The  U.  S.  Public  Health  Service  and  the  Bureau  of 
Chemistry  of  the  U.  S.  Department  of  Agriculture,  in-so-far  as  these 
methods  are  applicable. 

I.  Substances  to  be  analyzed. 

1.  Liquids  of  all  kinds. 

2.  Semiliquids  and  semisolids  miscible  with  water. 

3.  Solids  of  all  kinds. 

II.  Numerical  and  quantitative  limits  of  comtamination  in  different  substances. 

1.  For  mold — quantity  of  spores  and  hyphae. 

2.  For  yeasts — number  and  kind. 

3.  For  bacteria — number  and  kind. 

4.  For  pus,  dirt,  sand,  etc. 
Ill  Methods. 

1.  Making  concentrations. 

2.  Making  dilutions. 

3.  Making  the  counts  and  estimates, 
(a)  Bacteria. 

(6)  Yeasts. 

(c)  Mold  spores  and  mold  hyphae. 

(d)  Algae,  in  drinking  waters,  etc. 

(e)  Protozoa. 

(/)   Pus  cells,  in  milk,  etc. 
(g)  Dirt,  sand,  etc. 

4.  Plate  counts — Petri  dish  cultures. 

(a)  Culture  media  used. 

(b)  Optimum  temperature. 

(c)  Time  of  incubation. 
IV.  Qualitative  determinations. 

1.  Apparatus. 

2.  Culture  media. 

3.  Stains. 

4.  Special  methods. 

(a)  Colon  group  of  bacilli. 

(6)  Presumptive  colon  bacillus  test. 

(c)  Sewage  streptococci. 

(</)  Dysentery  bacilli  and  amoebae. 

(e)  Bacillus  typbosus. 

(/)  Paratyphoid  group. 

(g)  Cholera  vibrio. 

(h)  Yeasts. 

(i)  Molds. 

(i)  Animal  parasites. 

(k)  Larvae,  ovae,  etc. 


414  PHARMACEUTICAL  BACTERIOLOGY 

V.  Biological  water  analysis. 

1.  Bacteria,  number  and  kind. 

2.  Diatoms. 

3.  Desmids. 

4.  Nostoc. 

5.  Other  algae. 

6.  Molds;  significance  of. 

7.  Evidence  of  soil  and  sewage  contamination. 

VI.  Bacteriological  milk  analysis. 

1.  Quantitative. 

(a)  Standards  for  different  geographic  areas. 

(ft)  Summer  and  winter  standards — temperature  standards. 

2.  Qualitative. 

3.  Pus  and  blood  corpuscles;  significance  of. 

4.  Milk  diseases, 
(a)  Blue  milk 
(ft)  Ropy  milk. 

(c)   Bad  odors,  bad  taste,  etc. 

5.  Sour  milk. 

6.  "Buttermilk"  tablets. 

7.  Kefir,  koumys,  etc. 

VII.  Bacteriological  Examination  of  Shellfish. 

1.  Selection  of  sample. 

2.  Making  a  record  of  the  sample. 

3.  Transportation  of  the  sample. 

4.  Laboratory  procedure. 

5.  Bacterial  counts. 

6.  Determining  bacteria  of  the  colon  bacillus  group. 

7.  Statement  of  results.     Rating. 

VIII.  The  Bacteriological  and  Toxicological  Examination  of  Meat  and  Meat  Products. 

1.  Direct  microscopical  examination  of  meats. 
(a)  Bacteria  on  the  surface  of  meats, 

(ft)  Mold  and  mold  spores,  as  in  moldy  bacon,  pork,  fish,  etc 

(c)  Presence  of  bladder  worm,  larvae  of  parasites,  etc. 

(d)  Trichinae  in  pork  and  examination  for  trichinae. 

(e)  Cereal  fillers  and  starches  in  sausage  meats. 

(0   Preservatives  and  coloring  substances  in  meats. 

2.  Plate  cultures. 

(a)  Numerical  counts  of  bacteria. 

(ft)  Number  of  gas  formers  and  of  acid  formers. 

(c)   Bacillus  botulinus  in  pork  and  in  vegetables.     Botulism. 

3.  Toxicological  tests. 

(a)  Inoculation  tests  (guinea  pigs)  to  prove  the  absence  or  presence  of  toxins  or 

ptomaines, 
(ft)  Tests  for  tuberculous  meats  and  for  the  tubercle  bacillus. 

4.  Biological  Tests.     Determining  the  source  of  the  meat, 
(a)  Sugar  test  for  horse  meat. 

(ft)  The  precipitin  test  for  meats  from  different  animals. 

(c)   Microscopical  examination  of  tissues,  fats,  fat  crystals,  etc. 


MICROANALYTICAL   AND  BACTERIOLOGICAL   LABORATORY        415 

IX.  The  Bacteriological  Examination  of  Eggs  and  Egg  Products. 

1.  Direct  microscopical  examination. 
(a)  Bacteria. 

(6)  Molds. 

(c)  Mold  spores. 

2.  Plating  methods. 

3.  Egg  tests. 

(a)  Candling. 

(6)  Brine  test. 

(c)   Organoleptic  tests,  etc. 

4.  Evaporated  eggs. 

5.  Cold  storage  eggs,  etc. 

X.  Bacteriological  Examination  of  Pharmaceutical  Products. 

1.  Direct  microscopical  examination, 
(a)  Bacteria. 

(6)  Molds. 

(c)  Mold  spores. 

(d)  Yeasts. 

2.  Plating  methods. 

3.  Colon  bacillus  test. 

4.  Tetanus  bacillus  test. 

5.  Tests  for  the  staphylococcus  and  strep tococccus  groups. 

XI.  The  Microscopical  and  Bacteriological  Examination  of  Syrups. 

1.  Medicinal  syrups.  • 

(a)  Official,  simple  and  medicated. 

(6)  Patent  and  proprietary  medicated  syrups, 
(c)   Medicinal  preparations  containing  syrup. 

2.  Soda  fountain  syrups. 

3.  Fruit  juices  containing  sugar.     Fruit  juice  concentrates. 

4.  Syrups,  molasses,  treacle,  corn  syrup,  etc. 

XII.  The  Microscopical  and  Bacteriological  Examination  of  Fermented  Foods  and 
Drinks. 

1.  Whiskey  and  brandy. 

2.  Beer.     Beer  diseases. 

3.  Wines.     Wine  diseases. 

4.  Other  fermented  drinks. 

(a)  Sake  or  Japanese  rice  wine. 

(b)  Arrak. 

(c)  Yoghurt. 

(d)  Kephir. 

(e)  Koumiss. 
(/)    Soja  sauce, 
(g)  Mazun. 
(A)  Leban.  • 

(i)   Ginger  beer. 

(7)  Beebe  wine. 

XIII.  The  Bacteriological  Examination  of  Mineral  Waters. 

1.  Examination  of  centrifugalized  sediments. 

2.  Plating  methods. 

3.  Presumptive  colon  bacillus  test. 


416  PHARMACEUTICAL  BACTERIOLOGY 

XIV.  Determining  the  Efficiency  Value  of  Disinfectants. 

1.  Phenol  germ  destroying  coefficient. 

2.  Toxic  Coefficient. 

3.  Albumen  coagulating  coefficient. 

4.  Comparative  cost. 

XV.  Determining  the  Purity  and  Quality  of  Sera,  Bacterins  and  of  Related  Products. 

1.  Purity  and  freedom  from  bacteriological  contamination. 

2.  The  purity  of  smallpox  vaccines. 

3.  Purity  of  bacterial  vaccines. 

XVI.  Special  Biological  and  Toxicological  Tests. 

1.  Arsenic  in  foods.     Biological  test  for  arsenic. 

2.  Toxicity  tests  with  defibrinated  blood, 
(a)  Toxalbumins  and  toxins. 

(&)  Saponins. 

(c)   Chemical  hemolysis. 

3.  Frog  tests  for  the  presence  of  alkaloids. 

The  following  blank  will  be  found  useful  in  making  reports  of 
bacteriological  examinations.  In  many  instances  however,  it  will  be 
found  necessary  to  supplement  the  report  or  to  make  a  special  report. 

FORM  No.  Ill 
Bacteriological  Examination 

(No.,  label,  dates,  condition  of  seals  as  for  Form  I.) 

I.  Direct  count.     (Thoma-Zeiss  hemacytometer  with  Turck  ruling.) 

1.  Bacilli  per  cc 

2.  Cocci  per  cc 

II.  Plate  and  tube  cultures.     (Lactose-litmus-agar.) 

1.  Temperature  differential  test. 

(a)  (20°  C.)  colonies  per  cc '. 

(&)  (38°  C.)  colonies  per  cc 

2.  Color  differential  test. 

(a)  Pink  or  yellow  colonies  per  cc 

(6)  Not  pink  or  yellow  colonies  per  cc 

3.  Gelatine  liquefying  colonies  per  cc 

4.  Indol  reaction  ( ±) 

5.  Neutral  red  reduction  (±) 

6.  Gas  (hydrogen)  formula 

7.  Gram-stain  behavior  (  ±) 

8.  Presumptive  colon  bacillus  test  (  ±), 

(a)  Amounts  used 

(&)  Number  of  tests 

(c)   Rating 

III.  Special  tests 


IV.  Conclusions 

Analyst. 


MICROANALYTICAL  AND  BACTERIOLOGICAL  LABORATORY        417 

Course  III.  Sanitary  Science,  Including  Parasitology. — This 
course  is  to  be  given  during  the  fourth  year  of  the  full  four  year 
course  in  a  properly  equipped  college  of  pharmacy.  A  standard  text- 
book should  be  used,  supplemented  by  lectures,  laboratory  work  and 
practical  demonstrations,  and  extending  throughout  the  entire  college 
year.  Under  the  present  arrangements  of  the  college  curriculum  this 
course  must  be  given  concurrently  with  the  course  in  Food  Bacteri- 
ology (Course  II). 

V 

I.  Symbiology.     Parasitology. 

1.  Beneficent  symbioses. 
(a)  Mutualism. 

(&)  Individualism. 

2.  Commensalism.     Nutricism. 

3.  Antagonistic    symbioses.     Parasitism.     Parasitology    (exclusive    of  pathogenic 
bacteria). 

(a)  Exo-parasites — Lice,  etc. 
(6)  Skin  parasites — Itch,  etc. 

(c)  Intestinal  parasites — Tape  worm,  etc. 

(d)  Blood  parasites — Malaria,  etc. 

4.  Compound  symbioses.     Paracytoses.     Patrocytoses. 

II.  Sanitary  Rules,  Laws  and  Regulations. 

1.  National,  State  and  city. 

2.  Quarantineable  diseases. 

(a)  The  national  quarantine. 
(6)  The  state  quarantine. 

(c)  The  city  or  community  quarantine. 

(d)  The  family  or  house  quarantine. 

3.  Reportable  diseases. 

4.  Occupational  diseases. 

5.  Water  supplies  and  purification  of  drinking  water. 

6.  Sewage  disposal. 

7.  Disease  prevention. 

III.  Sterilization  and  Disinfection. 

1.  The  more  important  disinfectants. 

2.  Disinfection  and  fumigation, 
(a)  Public  buildings. 

(&)  Railway  disinfection. 

(c)  Street  car  disinfection. 

(d)  Private  dwellings. 

(e)  Sick  room  disinfection. 

IV.  Epidemiology. 

i.  The  more  important  pandemical,  epidemical  and  endemical  diseases, 
(a)  Influenza  or  La  Grippe. 
(&)  Asiatic  cholera. 

(c)  Bubonic  plague. 

(d)  Smallpox. 

(e}  Yellow  fever. 
(/)   Malaria. 
27 


41 8  PHARMACEUTICAL  BACTERIOLOGY 

(g)  Typhoid  fever. 
(A)  Diphtheria. 

2.  The  prevention  and  control  of  epidemics. 
(a)  Malarial  control. 
(&)  Typhoid  control. 

(c)  Smallpox  control. 

(d)  Plague  control. 

(e)  Yellow  fever  control. 
(/)   The  social  diseases. 

V.  Pure  Food  and  Drug  Laws,  Rules  and  Regulations. 

1.  National,  state  and  city. 

2.  Standards  of  quality  and  purity. 

3.  The  enforcement  of  the  laws. 

2.  The  Laboratory 

A.  Location  of  Laboratory.— It  may  be  in  a  separate  building,  as  the 
home,  but  as  a  rule  a  corner  room  in  the  pharmacy  is  best  suited  for  the 
purpose.     This  room  may  be  in  the  basement,  or  on  the  first,  second  or 
other  floor.     Do  not  select  a  room  with  a  through  passage  for  obvious 
reasons.     It  may  adjoin  a  chemical  or  pharmaceutical  laboratory,  though 
it  should  not  be  a  part  of  such  laboratories.     Chemicals  and  chemical 
fumes  interfere  with  bacteriological  and  microscopical  work.     It  should 
have  one  door  and  two  or  more  windows.     There  must  be  good  light  and 
the  environment  should  be  favorable  for  bacteriological  work,  for  which 
reason  a  room  in  the  basement  is  not,  as  a  rule,  desirable, 

The  walls  and  ceiling  of  this  room  should  be  absolutely  plain  and  well 
protected  by  white  enamel  paint.  The  floor  may  be  cement,  slate,  or 
hard  wood,  well  oiled  with  boiled  linseed  oil,  or  it  may  be  painted,  or 
covered  with. linoleum.  The  entire  room  (walls,  ceiling,  floor)  should 
be  washed,  scrubbed  and  disinfected  from  time  to  time.  That  is,  it 
should  be  kept  bacteriologically  clean. 

B.  Furnishings. — All  windows  exposed  to  direct  sunlight  should  have 
white   translucent  roller  shades.     The  laboratory  should  be  well  sup- 
plied with  gas;  water,  both  hot  and  cold;  and  means  for  lighting  (gas, 
electricity,    acetylene).     There    should   be   just   enough   furniture  and 
shelving,  no  more.     One  table  with  slate  top  or  lined  with  linoleum;  one 
stool,  shelves  for  samples,  apparatus  and  reagents.     A  case  for  chemicals, 
cotton,  culture  media,  etc.     A  case,  with  lock  and  key,  for  samples  to  be 
examined.     The  plumbing  must  be  of  the  best  and  the  fixtures  must  be  of 
safe  construction.     The  sink  should  be  large  and  deep  and  should  be  lined 
with  porcelain  and  supplied  with  an  ample  drain  board.    A  hood  or  ventila- 
tor should  be  provided  to  carry  off  steam  vapors.     Near  the  table  for 
microscopical  work  should  be  a  shelf  or  case  for  the  works  of  reference. 

C.  Apparatus. — There  will  be  required: 
(a)  A  good  simple  lens. 


MICROANALYTICAL   AND   BACTERIOLOGICAL   LABORATORY         419 

(b)  Compound  microscope. 
Ocular  with  micrometer  scale. 
Oculars,  Nos.  2  and  3. 

Objectives,  Nos.  3,  5,  7  and  ^{2  in.  oil  immersion. 

(c)  Slides  and  covers. 

(d)  Section  knife  or  razor,  and  strop. 

(e)  Polarizer,  for  the  study  of  starches,  crystals,  etc.  Should  be  conven- 
ient to  use.     This  is  very  important.     The  selenite  plates  which  are 
usually  supplied  with  the  polarizer  are  useful. 

(/)  Thoma-Zeiss  hemacytometer  with  Turck  ruling,  for  counting  bacteria 
spores  and  yeast  cells  in  vinegar,  jams,  jellies  and  other  like  substances. 
Other  special  counting  chambers. 

(g)  Accurately  ruled  metal  or  hard  rubber  millimeter  ruler  for  measuring 
seeds  in  fruit  products,  etc. 

(h)  One  Arnold  steam  sterilizer  (copper).  A  vegetable  steam  cooker 
will  serve. 

(i)  One  hot  air  sterilizer.  The  ordinary  double  walled  baking  ovens 
which  may  be  secured  from  any  hardware  dealer,  will  serve  the  purpose. 
Cut  in  a  small  opening  at  top  for  the  thermometer. 

(/)  One  rice  cooker  in  which  to  prepare  culture  media,  etc. 

(k)  One  small  incubator  with  thermo-regulator. 

(/)  Centrifuge  (electric). 

In  addition  to  the  above  there  will  be  required  the  necessary  chemicals, 
reagents,  etc.,  good  quality  commercial  cotton  for  plugging  test-tubes, 
medium  size  Petri  dishes,  flasks  (J/£  liter  and  i  liter),  several  evaporating 
dishes,  one  or  two  moist  chambers,  a  quantity  of  medium  size  test-tubes, 
slide  boxes,  test-tube  brushes,  dissecting  needles,  scalpels,  labels,  pencils, 
etc.  Get  the  necessary  things  only.  There  must  be  a  liberal  supply  of 
clean  towels.  No  one  but  the  analyst  and  his  assistants  should  have 
access  to  the  laboratory.  On  entering,  the  analyst  should  remove  coat 
and  hat  and  put  on  a  white  clean  linen  apron  and  coat,  such  as  are  worn  by 
soda  fountain  dispensers.  This  white  suit  should  remain  in  the  laboratory 
and  should  be  changed  for  a  clea;i  one  as  often  as  may  be  necessary. 

Special  equipment  and  apparatus  may  be  indicated  as  the  work  pro- 
gresses. For  instance,  it  may  prove  desirable  to  have  an  incubator  for 
opsonic  work,  for  the  use  of  physicians,  used  either  by  the  physicians 
or  by  the  pharmacist.  A  water  filtering  equipment  may  be  installed, 
likewise  a  water  still.  An  autoclave  may  prove  desirable.  There  are 
matters  which  must  be  left  to  the  individual  pharmacist.  The  following 
is  an  outline  of  such  work  as  the  pharmacist  may  do  in  the  microscopical 
and  bacteriological  laboratory. 


42O  PHARMACEUTICAL  BACTERIOLOGY 

D.  Micro-analytical  Work. — The  practical  work  which  may  be  done  will 
depend  upon  the  special  preparation  and  qualification  of  the  pharmacist, 
as  has  been  indicated  in  the  preceding  pages,  and  also  upon  the  opportuni- 
ties which  may  offer  themselves.  The  following  is  a  partial  list  of  sub- 
stances which  may  be  examined  bacteriologically  or  microscopically,  or 
both.  Other  work  is  also  indicated. 

(a)  Vegetable  drugs,  crude  and  powdered. 

(0)  Spices  and  condiments,  whole,  ground  and  powdered.     Prepared 
spices  and  condiments. 

(c)  Coffee,  tea,  cocoa,  chocolate,  confections,  candies. 

(d)  Tobacco,  including  smoking  tobacco,  cigars,  cigarettes,  snuff. 

(e)  Compound  powders,  pharmacopoeial  and  others.  . 
(/)  Tablets,  pills,  simple  powders. 

(g)  Meats;  raw,  cooked,  canned,  sausage  meats,  mince  meats,  etc. 
(ti)  Dairying  products  as  milk,  cream,  cheese,  butter,  ice  creams, 
cream  fillers. 

(1)  Cosmetics,  dusting  powders,  insect  powders. 

(f)  Cattle  and  poultry  powders. 

(k)  Starches,  dextrins,  sausage  meat  binders  (starchy). 
(/)   Vegetable  foods;  as  jams,  jellies,  fresh,  pickled,  cooked,  canned 
and  preserved. 

(m)  Flours  and  meals. 

(ri)  Breakfast  foods,  baby  food,  invalid  foods. 

(0)  Breads,  cakes,  pies,  crackers,  etc. 

(/>)  Catsups,  tomato  pastes,  etc. 

(g)  Macaroni,  spaghetti,  noodles,  etc. 
(r)  Nuts,  and  nut-like  fruits. 

(s)   Cloth  material,  textile  fabrics  generally,  cordage,  papers,  etc. 

It  is  assumed  that  the  pharmacist  has  had  the  necessary  training  to 
undertake  the  microscopical  examination  of  the  substances  above  classi- 
fied, with  the  aid  of  such  standard  works  of  reference  as  may  be  required. 
The  micro-analyses  should  also  include: 

1.  Gross  and  net  weight  determination  of  all  samples  that  require  it, 
for  which  purpose  an  accurate  balance  is  necessary. 

2.  Moisture  determinations  of  such  substances  as  may  require  it. 
There  should  be  no  difficulty  in  constructing  the  necessary  apparatus  for 
making  moisture  determinations. 

3.  Ash  determinations  of  substances  which  require  it.     This  calls  for 
a  special  equipment  including  a  platinum  dish,  ignition  furnace  with 
burners,  etc. 

4.  Use  of  special  tests,  as  sublimation  tests  for  benzoic  acid  and  sali- 
cylic acid,  the  hand  wheat  gluten  test,  Bamihl  gluten  test,  Grahe's  cin- 


MICRO  ANALYTICAL  AND  BACTERIOLOGICAL   LABORATORY        421 

chona  test,  color  reaction  tests  for  boric  acid,  salicylic  acid,  morphine,  and 
opium;  tests  for  phytosterol  and  cholesterol  crystals,  etc.,  etc.  These 
and  other  tests  are  explained  in  the  several  reference  works  cited  above. 
In  the  examination  of  liquids  or  semi-liquids  as  wines,  beers,  cider,  vine- 
gar, milk,  cream,  sewage,  extracts,  tinctures,  etc.,  a  centrifuge  is 
desirable. 

E.  Bacteriological  Work. — The  pharmacist  should  be  prepared  to  dp 
the  following  work  in  the  bacteriological  laboratory. 

(a)  Prepare  culture  media  for  use  of  physicians,  as  may  be  required. 

(b)  Prepare  sterile  throat  swabs  for  the  use  of  physicians. 

(c)  Prepare  stains  and  do  staining  for  physicians,  as  may  be  required. 

(d)  Make  bacteriological  determinations  of  milk,  jams,  jellies,  im- 
pure drinking  water,  vinegar,  wine,  sera,  vaccines,  antitoxins,  contam- 
iated  foods  and  drinks,  sewage,  etc. 

(e)  Sterilize  pharmaceuticals,  surgical  supplies,  etc. 

(/)  Assist  the  physician  in  opsonic  work,  as  may  be  arranged  or  agreed 
upon. 

(g)  Do  bacterial  culture  incubation  work  for  the  physician,  make  sub- 
cultures, Wassermann  test  for  syphilis,  etc. 

(h)  Filter  and  sterilize  drinking  water  to  be  supplied  to  customers. 

F.  The  Cabinet  of  Microscopic  Exibits. — In  every  laboratory  where 
bacteriological  and  microanalytical  work  is  being  done,  there  should  be 
an  exhibit  of  all  of  the  substances  which  are  likely  to  come  under  obser- 
vation, in  order  that  any  desirable  comparison  may  be  immediately 
made.    The  following  suggestions  are  for  the  cabinet  intended  for  general 
microanalytical  work.    Those  interested  can  readily  prepare  a  special, 
more  limited  cabinet,  from  the  suggestions  hereby  presented. 

The  exhibit  is  to  consist  of  objects  and  materials  which  may  prove 
of  analytical  value  and  use  in  making  .comparisons  and  for  purposes  of 
check  work  and  re-verification.  The  exhibit  is  not  to  include  samples  of 
the  materials  secured  for  the  purpose  of  study  or  examination  as  to 
identity,  quality  or  purity.  Such  materials  may  be  kept  in  a  separate 
cabinet,  and  for  such  periods  of  time  only,  as  they  may  or  might  be  of  use 
for  further  study  and  comparison.  If  the  examination  shows  that  the 
article  is  of  the  quality  for  which  it  was  purchased,  then  no  sample  need 
be  kept.  If  it  proved  to  be  adulterated  or  of  inferior  quality,  then  a 
sample  should  be  retained  until  the  matter  is  finally  settled  or  disposed  of. 
In  case  of  a  retail  pharmacy,  the  stock  of  drugs  on  hand  is  the  exhibit 
of  the  articles  which  have  been  examined  and  compared  with  the  articles 
in  the  microscopic  cabinet.  Should  the  microanalyst  devote  his  entire 
effort  to  food  products,  then  the  cabinet  will  be  stocked  with  pure  and 
representative  food  products  spices,  flours,  meals,  etc. 


422  PHARMACEUTICAL  BACTERIOLOGY 

The  greatest  care  must  be  observed  in  the  preparation  of  the  micro- 
scopic cabinet,  particularly  as  to  the  identity  of  the  samples  and  the  labels 
attached  thereto.  Nothing  is  to  be  included  of  which  the  source  is  in 
any  way  questionable.  All  samples  must  be  obtained  from  absolutely 
reliable  sources  and  even  then  each  and  every  sample  must  be  carefully 
examined  in  order  to  make  absolutely  certain  that  it  is  genuine.  Drug 
samples  must  be  secured  from  reliable  dealers,  food  and  spice  samples 
from  reliable  merchants;  samples  of  cloth,  of  furs,  of  paper,  of  cordage, 
etc.,  from  specialists  in  the  several  products.  The  statements  of  the  in- 
experienced lay  man  must  not  be  accepted.  To  illustrate,  the  owner  of  a 
rug  may  emphatically  declare  it  to  be  a  genuine  Bokhara,  basing  this  em- 
phatic declaration  upon  the  misstatements  of  an  unscrupulous  dealer.  A 
sample  of  ground  black  pepper  may  be  declared  of  prime  grade  or  quality 
by  an  experienced  dealer  hi  spices,  whereas  it  may  be  made  of  "grinding 
peppers."  In  the  case  of  articles  which  are  believed  to  be  of  special  im- 
portance, the  source  or  identity  of  which  is  not  clearly  known,  a  commer- 
cially non-prejudiced  and  non-interested  expert,  or  several  such  experts, 
should  be  consulted.  If  the  article  cannot  be  identified  for  a  certainity, 
it  must  be  discarded  and  may  not  be  used  for  purposes  of  comparison. 
It  may  be  filed  in  a  special  case  for  samples  of  this  kind,  with  the  hope 
that  its  exact  identity  may  at  some  future  time,  be  ascertained.  The 
following  are  suggestions  for  the  formation  of  a  general  microanalytical 
exhibit. 

It  is  most  important  to  guard  against  any  excess  in  the  size  or  bulk 
of  the  exhibit;  the  smallest  quantities  in  the  most  compact  groups,  should 
be  the  guide.  Use  the  smallest  containers  which  will  serve  the  purpose, 
and  have  them  of  uniform  size  in  so  far  as  possible  and  practicable.  The 
containers  must  be  easily  accessible,  the  caps,  stoppers  or  other  sealing, 
readily  removable  and  replaceable,  without  danger  of  becoming  mixed 
or  misplaced  or  displaced.  Too  much  care  cannot  be  given  to  the  label- 
ing. The  legends  thereon  must  be  distinct  and  legible  and  sufficiently 
full  and  concise,  so  that  the  intelligent  observer  may  at  once  know  the 
meaning  or  full  significance.  On  the  other  hand,  meaningless  and  wholly 
useless  and  mentally  confusing  details  must  be  omitted  from  the  labels. 

i.  Crude  and  Powdered  or  Ground  Samples.  Bulk  Samples. — A 
hard  wood  cabinet,  similar  to  those  which  are  in  use  in  food  and  drug 
laboratories  for  holding  samples  of  pure  foods,  spices,  vegetable  drugs, 
fiber,  powders,  chemicals,  etc.,  will  serve  the  purpose  excellently.  The 
containers  are  usually  of  glass,  with  screw  tops,  and  rest  horizontally  in 
suitable  hollow  grooves  of  the  drawers.  Small  rubber  stoppered  or  cork 
stoppered  Homeopathic  vials  are  excellent,  if  the  regulation  containers  are 
not  available.  The  cabinet  should  hold  several  thousand  articles,  each 


MICROANALYTICAL  AND  BACTERIOLOGICAL   LABORATORY        423 

and  every  one  distinctly  labelled.  Containers  with  liquids  generally,  and 
oils,  should  not  be  placed  horizontally,  but  vertically,  and  the  sealing 
must  be  perfect,  for  reasons  which  are  self-evident.  The  samples  must 
be  representative  and  hi  many  instances  they  must  be  reduced  and  com- 
minuted so  as  to  get  them  into  the  containers.  A  few  samples  may  re- 
quire the  use  of  wide-mouthed  containers,  such  as  the  ounce  quinine  vials. 
Not  more  of  these  should  be  used  than  is  absolutely  necessary,  as  they 
take  up  too  much  space.  Many  of  the  articles  may  be  filed  away  in  book 
form,  or  pasted  in  book  form,  or  placed  in  small  envelopes  which  in  turn 
are  pasted  in  blank  books  which  are  equal  in  size  and  form  to  the  other 
sample  books.  The  substances  to  be  placed  in  the  cabinet  may  be  grouped 
as  follows: 

a.  For  the  containers  which  are  horizontally  placed;  solids  and  powders 
of  all  kinds,  broken,  cut  and  trimmed  substances.     Gums,  resins,  waxes, 
some  pastes  and  other  semisolids.    Solid  chemicals,  most  metals,  etc. 

b.  For  the  containers  which  are  to  be  placed  vertically;  liquids  gener- 
ally, oils,  syrups,  chemicals  in  solution,  etc. 

c.  For  the  books  all  of  uniform  size;  papers  of  all  kinds,  cloth  of  all 
kinds,  cordage,  animal  hair,  samples  of  furs,  vegetable  fiber  generally,  etc. 

These  physical  groups  may  be  made  to  include  foods  of  all  kinds  ex- 
cepting those  which  it  is  not  necessary  or  desirable  to  keep  on  hand,  drugs, 
poisons,  narcotics,  cloth,  silks,  metals,  minerals,  etc.  It  is  impracticable 
and  usually  wholly  unnecessary  to  preserve  the  readily  perishable  articles, 
such  as  fresh  meats,  fresh  vegetables,  cheese,  bread  and  pastries.  All 
materials  in  the  cabinet  must  be  non-decomposable,  either  naturally  so 
or  rendered  so  through  the  addition  of  some  suitable  preservative.  Some 
food  substances  may  be  artificially  desiccated  before  placing  them  into 
the^  containers. 

The  sample  books  are  to  be  placed  in  the  bottom  drawer  of  the  cabinet, 
in  suitable  compartments,  and  may  include  the  following: 

(a)  Books  of  samples  of  commercial  papeis  (book  paper,  note  paper, 
writing  paper,  etc.) 

(b)  Books  of  wrapping  paper,  tissue  paper,  newspaper,  etc. 

(c)  Book  of  felt  papers  of  all  kinds. 

(d)  Book  of  filter  papers  of  all  kinds. 

(e)  Books  of  cloth  of  all  kinds. 

(/)  Books  of  cordage,  threads,  twines,  etc. 

(g)  Book  of  parchments,  bank  notes,  paper  currency,  etc. 

(h)  Book  of  samples  of  furs  of  all  kinds. 

(i)  Book  of  silk  samples,  natural  as  well  as  artificial. 

(/')  Book  of  miscellaneous  fiber  and  cloth  material,  etc. 

Most  of  the  books  mentioned  may  be  obtained  from  the  special  dealers 


424 


PHARMACEUTICAL  BACTERIOLOGY 


and  manufacturers,  but  they  do  not  come  in  uniform  sizes.  They  may  be 
trimmed  to  uniform  size  and  the  materials  rearranged,  or  the  articles 
may  be  removed  and  repasted  into  the  blank  books  of  uniform  size.  Such 
work  must  be  very  carefully  done  to  avoid  mistakes  and  confusion  in  the 
placing  of  labels  and  the  descriptions. 


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FIG.  96. — Plan  for  a  food  and  drug  exhibit  cabinet.  The  outside  dimensions  are 
four  feet  high,  two  and  one-half  feet  wide,  and  two  feet  deep.  It  is  to  be  made  of  well 
seasoned  hard  wood,  and  all  of  the  drawers  must  work  easily  and  smoothly  and  so 
fastened  that  they  will  not  drop  out  when  drawn  to  full  distance.  A  cabinet  of  the 
above  dimensions  can  be  made  to  contain  sixteen  shallow  drawers  for  the  horizontally 
placed  screw  top  containers,  each  drawer  holding  120  such  containers  placed  in  tiers 
and  lying  in  suitable  grooves;  two  drawers  for  the  vertically  placed  containers  for  the 
liquid  substances  which  are  set  in  rows  in  holes  in  a  deck  shelf  and  a  shallow  base  well; 
and  the  large  bottom  drawer  for  the  exhibits  in  book  form.  The  drawers  may  be 
lettered  a,  b,  c,  etc.,  and  the  articles  in  each  drawer  numbered  seriatim  i,  2,  3,  etc.; 
thus,  "  Oil,  whale, "  might  be  located  by  "#-143."  The  case  may  be  provided  with  lock 
and  key. 

The  above  articles  may  be  cared  for  in  a  cabinet,  the  outside  measure- 
ments of  which  do  not  exceed  4  feet  in  height,  2%  feet  in  width  and  2  feet 
in  depth.  Such  a  cabinet  may  have  sixteen  shallow  drawers  for  the  hori- 
zontally placed  containers  (about  1,000  containers);  two  drawers  five 
inches  deep  for  the  liquids  (about  500  samples) ;  and  a  single  deep  bottom 
drawer  for  the  sample  books  placed  on  edge,  suitably  grouped  in  second- 
ary compartments  (altogether  containing  from  1,000  to  2,000  samples). 

The  articles  in  the  cabinet  above  described  are  for  microscopical  exami- 
nation, as  has  been  indicated,  but  before  this  can  be  done,  each  and  every 


MICROANALYTICAL    AND   BACTERIOLOGICAL    LABORATORY       425 

substance  must  be  properly  mounted  upon  a  slide.  There  are  many  sub- 
stances which  it  is  convenient  to  have  on  hand  ready  mounted  for  imme- 
diate microscopic  examination.  These  are  to  be  kept  in  a  second  smaller 
cabinet,  known  as  the  cabinet  for  microscopic  slides. 

2.  Cabinets  of  Microscope  Slides. — The  cabinets  of  various  sizes  can  be 
obtained  from  dealers  in  microscopic  supplies  and  are  intended  to  hold 
slide  mounts  for  microscopic  examination.    The  slide  mounts  are  ob- 
tained from  various  sources.     Many  will  be  prepared  by  the  analyst, 
from  times  to  time.    Others  will  be  secured  from  dealers  in  scientific 
instruments  and  supplies  who  generally  furnish  lists  of  the  slides  which 
they  offer  for  sale,  more  especially  representing  the  following  groups  of 
organisms  and  tissues. 

(a)  Bacteria.     Molds  and  other  fungi.     Protozoa. 

(b)  Desmids,  diatoms,  and  other  low  forms  of  algae. 

(c)  Embryological  slides. 

(d)  Vegetable  tissues  of  all  kinds. 

(e)  Animal  tissues,  normal  and  pathological. 
(/)  Crystals,  minerals,  earths,  etc. 

The  State  and  Local  Boards  of  Health  may  furnish  slides  of  the  principle 
disease  germs,  etc.,  free  for  demonstration  purposes,  and  such  a  cabinet 
should  be  added  to  the  exhibit.  The  ready  prepared  sets  of  slides 
should  be  carefully  selected  so  as  not  to  encumber  the  exhibits  with 
things  of  little  or  not  practical  use.  The  slides  will  be  of  inestimable 
value  in  some  of  the  analytical  work.  One  medium  size  slide  cabinet 
will  be  sufficient  as  a  beginning.  A  complete  index  to  the  slides  must 
be  prepared  and  such  index  must  be  conveniently  usable. 

3.  Photo-micrographs. — There  should  be  a  photo-micrograph  of  each 
and  every  article  mounted  upon  a  microscope  slide.    The  prints  should 
be  mounted  upon  suitable  cards  and  every  photograph  should  be  very 
carefully  and  accurately  labelled.     It  would  be  desirable  to  have  two 
sets  of  these  photographs.    One  set  in  a  filing  case,  in  alphabetical  se- 
quence according  to  common  English  names.    The  other  set  pasted  into 
a  book,  in  groups  following  the  appended  outline.    The  book  would  be 
very  desirable  for  quick  reference  and  for  comparative  demonstration 
purposes. 

It  is  suggested  that  the  articles  in  the  three  divisions  of  the  exhibit, 
above  explained,  should  be  arranged  in  alphabetical  sequence  according 
to  well  established  and  generally  recognized  common  English  names,  ex- 
cepting that  a  certain  amount  of  special  grouping  is  desirable  or  neces- 
sary, as  already  explained.  As  the  exhibit  gains  in  size  it  may  become 
necessary  to  adopt  some  other  system  of  arrangement. 

For  the  benefit  of  those  who  are  entrusted  with  the  building  up  of  an 


426  PHARMACEUTICAL   BACTERIOLOGY 

exhibit  of  the  kind  above  specified,  the  following  brief  instructions  in 
making  microscopic  mounts,  are  given. 

i.  Smears. — Slide  mounts  for  microscopica1  examination,  generally 
known  as  smeais,  may  be  made  fiom  a  great  variety  of  substances,  more 
especially  from  liquid,  semiliquid  and  pasty  mateiials,  including  blood, 
sputum,  pus,  urinary  sediment,  feces,  excretions  and  secretions,  bacteria 
and  bacterial  cultures  and  all  substances  containing  bacteria,  etc. 
Temporary  smears  may  be  made  of  fats,  butter,  oleomargarine,  oils,  cieam, 
and  oily  substances  generally.  Permanent  smear  slides  intended  foi  the 
exhibit  aie  to  be  made  as  follows.  Clean  a  slide  very  carefully  by  means 
of  alcohol  and  a  clean  cloth,  passing  it  through  the  flame  of  a  Bunsen 
burner  after  the  wiping.  Spread  the  substance  very  thinly  and  evenly 
upon  the  middle  portion  of  the  cleaned  slide  and  allow  it  to  air  dry  (very 
moderate  heat  may  be  used,  not  to  exceed  7o°C.).  Next  "fix"  the  smear 
upon  the  slide  by  passing  it  through  the  flame  of  the  Bunsen  burner  four 
or  five  times,  or,  by  adding  a  drop  of  alcohol.  The  heat  (or  the  alcohol) 
coagulates  the  albuminous  matter  which  may  be  present,  and  sticks  the 
objects  firmly  to  the  surface  of  the  slide. 

The  air  dried  and  "fixed"  smear  may  now  be  examined  and  if  the 
mount  proves  satisfactory  (spread  sufficiently  thin  to  make  details  dis- 
tinct, etc.),  it  may  be  labeled  and  filed  away.  No  cover  glass  or  mounting 
medium  is  used.  Or,  it  may  be  stained  by  means  of  methyl ene  blue, 
LoefHer's  methylene  violet,  Fuchsin,  Safranin,  Bismarck  brown,  Wright's 
stain,  Giemsa's  stain,  etc.  The  selection  of  the  stain  will  depend  upon  the 
object  it  is  desired  to  attain.  The  staining  should  be  omitted  until  it  is 
known  for  a  certainty  that  staining  is  desirable  and  the  operator  knows 
how  to  use  it.  The  unstained  mounts  will  keep  indefinitely  and  may  be 
stained  any  time.  The  only  difficulty  lies  in  the  fact  that  since  the  smear 
mounts  are  to  be  examined  by  means  of  the  3^2  oil  immersion  lens,  the 
oil  used  (cedar  oil)  would  interfere  more  or  less  with  the  staining. 

A  blood  smear  requires  special  manipulation.  Place  a  droplet  of  the 
blood  (or  the  liquid  containing  the  blood)  upon  a  thoroughly  cleaned  slide, 
nearer  one  end.  Now  place  one  end  of  a  second  cleaned  slide  just  beyond 
the  droplet,  at  an  angle  of  30°— 40°,  lower  the  end  until  the  surface  of  the 
tilted  .slide  comes  in  contact  with  the  droplet,  and  then  gently  draw  it 
forward  over  about  %  of  the  slide  surface  (that  is,  the  slide  holding  the 
droplet).  Another  method  for  spreading  is  as  follows.  Place  the  second 
tilted  slide  on  the  other  side  of  the  droplet,  lower  until  it  comes  hi  capil- 
lary contact  with  the  droplet  and  then  push  it  over  the  surface  of  the  first 
slide,  instead  of  drawing  it  forward,  as  in  the  first  method.  After  spread- 
ing, the  smear  is  allowed  to  air  dry  and  then  fixed  by  means  of  a  drop  of 
alcohol,  instead  of  heat.  The  stains  generally  used  for  blood  smears  are 


MICRO  ANALYTICAL    AND   BACTERIOLOGICAL    LABORATORY        427 

Wrigth's  and  Giemsa's.     (Consult  some  modern  work  on  Bacteriology  or 
Parasitology  as  to  the  methods  for  preparing  any  of  the  desired  stains). 

2.  Balsam   Mounts. — Canada  balsam,   suitably   diltued  with  xylol, 
benzol,  oil  of  cloves,  ether,  etc.,  is  used  for  making  permanent  mounts. 
The  balsam  should  be  diluted  to  the  consistence  of  thin  syrup,  or  rather 
thin  oil.     In  fact  it  should  be  thin  enough  so  that  when  a  droplet  is  placed 
near  the  edge  of  a  cover  glass  upon  a  slide,  capillarity  should  draw  it  under 
and   spread  it  evenly.    Xylol  is  one  of  the  best  diluents  for  Canada 
balsam.     Oil  of  cloves  is  objectionable  because  it  destroys  more  or  less 
of  the  color  of  the  stained  mounts.     Otherwise  it  is  excellent. 

The  thing  of  special  importance  is  to  remember  that  all  substances 
which  are  to  be  mounted  in  Canada  balsam  must  be  entirely  free  from 
moisture:  if  any  considerable  moisture  is  present  the  mounts  become 
worthless,  because  the  water  will  not  mix  with  the  balsam,  forming  opaque 
emulsions.  All  substances  which  contain  moisture  may  be  dehydrated 
by  placing  them  in  alcohol  for  a  short  time;  until  the  water  has  become 
diffused  into  the  alcohol  and  evaporated.  After  dehydrating,  the  material 
should  be  placed  into  xylol  (or  the  same  substance  in  which  the  balsam  is 
dissolved)  for  a  few  seconds  and  then  mounted  in  the  balsam.  Balsam 
mounts  are  of  course  permanent. 

3.  Glycerine  Mounts. — Glycerine,  or  equal   parts    of   glycerine    and 
water,  makes  an  excellent  mounting  medium  and  has  the  advantage  over 
balsam  in  that  moisture  and  water  does  not  interfere  with  its  use.     Such 
mounts  will  keep  for  months  and  even  for  longer  periods  of  time,  but 
require  careful  handling,  as  they  are  easily  removed  from  the  slides.     If 
care  is  observed  in  mounting  (using  just  enough  of  the  mounting  medium) 
and  placing  the  cover  glass  carefully)  and  the  mounts  are  placed  in  suit- 
able slide  boxes,  they  may  be  kept  indefinitely.     The  glycerine  does  not 
evaporate,  or  if  it  does  vanish  partially  with  time,  more  may  readily 
be  added. 

Balsam  mounts  as  well  as  glycerine  mounts  require  the  use  of  cover 
glasses.  The  operator  should  bear  in  mind  the  effects  produced  by  the 
mounting  processes.  Heating  and  dehydrating  causes  a  reduction  in 
size  and  some  slight  distortion  in  form.  The  reduction  in  size  may  be 
considerable,  a  fact  which  must  be  kept  in  mind  when  comparing  one  and 
the  same  organic  substance  examined  in  the  fresh  state  and  in  the  per- 
manent mounts. 

A  cabinet  may  be  perfect  in  arrangement  and  it  may  be  completely 
stocked  and  each  and  every  article  contained  therein  may  be  fully  and 
accurately  labeled,  and  yet  such  a  cabinet  would  be  of  little  practical  use 
is  there  is  no  way  of  locating  the  articles  therein  contained.  Just  as  it 
should  be  made  a  criminal  offence  to  write  a  book  (especially  a  book  of 


428  PHARMACEUTICAL  BACTERIOLOGY 

science)  without  a  complete  index,  just  so  should  it  be  made  a  criminal 
offence  to  prepare  an  exhibbit  of  the  kind  above  explained  without  a 
complete  index.  In  the  matter  of  indices,  it  may  be  explained  that  some 
are  excellent  and  some  are  so  incomplete  as  to  be  of  little  value. 

For  the  purpose  of  the  above  exhibit,  the  following  classification  as  a 
basis  for  preparing  the  complete  list  of  articles,  is  suggested.  To  the 
right  of  each  group  and  each  individual  article,  is  given  the  number  of  the 
drawer  and  the  series  in  the  drawer  in  which  the  article  is  placed. 

Groups  of  the  Articles  of  the  Exhibit 

I.  Bacteria. 
II.  Beverages. 

1.  Alcoholic. 

2.  Non-alcoholic. 

III.  Blood. 

1.  Normal  (human). 

2.  Pathological  (human). 

3.  Animal  (normal  and  pathological). 

IV.  Chemicals. 

1.  Solid. 

2.  Liquid  and  solutes. 
V.  Cloth. 

1.  Animal. 

2.  Vegetable. 

3.  Mixed. 

VI.  Cordage  and  Thread. 
VII.  Earths,     (see  soils) 
VIII.  Fiber,  (not  woven) 

1.  Animal. 

2.  Vegetable. 

3.  Mineral. 

IX.  Flours  and  Meals. 
X.  Foods. 

1.  Animal. 

2.  Vegetable. 

3.  Mixed. 

XI.  Inks  and  Pigments. 
XII.  Minerals  (uncombined  elements) . 

XIII.  Parasites  (non-bacterial  and  non-protozoal) . 

XIV.  Protozoa  (parasitic  and  non-parasitic) . 
XV.  Secretions  and  excretions. 

1.  Normal. 

2.  Pathologic. 
XVI.  Soils. 

XVII.  Starches. 
XVIII.  Tissues  and  Tissue  Elements,  (animal  hair  not  included). 

1.  Animal. 

2.  Vegetable. 


MICRO  ANALYTICAL   AND  BACTERIOLOGICAL  LABORATORY       429 

It  will  be  found  that  everything  which  it  may  be  desired  to  file  may  be 
classified  under  one  or  the  other  of  the  groups  above  named.  In  so  far 
as  practicable,  every  article  should  be  listed  under  its  more  common 
English  name,  with  exceptions.  For  example,  the  various  slides  of  the 
group  bacteria,  may  be  arranged  in  the  alphabetical  sequence  of  the 
true  scientific  names.  A  like  classification  suggests  itself  for  the  groups 
protozoa  and  perhaps  parasites.  0 

A  properly  arranged  and  well  stocked  exhibit,  as  above  suggested,  is 
of  equal  importance  with  the  laboratory  equipment  and  the  working 
reference  library.  The  entire  exhibit  should  be  gone  over  carefully  from 
time  to  time  to  see  if  labels  are  still  in  place,  if  still  complete  according  to 
the  full  alphabetical  list,  etc.  Substances  which  have  undergone  de- 
composition or  other  spoilage,  should  be  discarded  and  replacedjby  fresh 
samples.  Leakage  must  be  corrected. 

The  following  is  a  diagram  of  a  bacteriological  and  microscopical 
laboratory: 


B 


PIG.  97. — Plan  of  bacteriological  and  microscopical  laboratory,  using  corner  room 
in  the  pharmacy.  (Scale  4  feet  to  the  inch.)  A,  shelves  on  three  sides  of  the  room, 
B,  work  table.  C,  cases  and  shelves  for  reagents,  chemicals,  glassware,  etc.  D,  case 
for  reference  books.  E,  sink.  F,  drain  board.  I,  Arnold  steam  sterilizer;  2  hot  waste 
filter;  3,  hot  air  sterilizer;  4,  rice  cooker;  5,  opsonic  incubator;  6,  incubator;  7,  compound 
microscope;  8,  stool;  9,  hat  and  coat  books. 


The  following  books  will  make  an  excellent  nucleus  to  which  additions 
are  to  be  made  from  time  to  time. 

i.  Bailey,  E.H.S.— The  Source,  Chemistry  and  Use  of  Food  Products. 
P.  Blakiston's  Son  and  Company.     1914. 


430  PHARMACEUTICAL   BACTERIOLOGY 

2.  Clayton,    Edw.    Godwin. — A  Compendium  of  Food  Microscopy. 
William  Wood  and  Company.     1908. 

3.  Herms,  William  B. — Medical  and  Veterinary  Entomology.     The 
MacMillan  Company.     1915. 

4.  Marshall,     Charles,     ( and     Collaborators )  . — Microbiology.      P. 
Blakiston's  Son  and  Company.     1912. 

5.  McCaughy,  W.  J.  and  Fry,  W.  R. — The  Microscopic  Determination 
of  Soil  Forming  Minerals.   *Bull.  No.  91  (March  25,  1913).     Bureau  of 
Soils,  U.  S.  Dept.     Agr. 

6.  Prescott,  Samuel  Gate. — Elements  of  Water  Bacteriology.     John 
Wiley  and  Sons.     1913. 

7.  Pryor,   James   Chambers. — Naval  Hygiene.     P.   Blakiston's  Son 
and  Company.     1918. 

8.  Rosenau,    Milton    J. — Preventive    Medicine    and    Hygiene.     D. 
Appleton  and  Company.     1917. 

9.  Schneider,  Albert. — Bacteriological  Methods  in  Food  and  Drug 
Laboratories.     P.  Blakiston's  Son  and  Company.     1915. 

10.  Schneider.   Albert. — The    Microanalysis  of  Powdered  Vegetable 
Drugs.     P.  Blakiston's  Son  and  Company.     1920. 

11.  Schneider,  Albert. — The  Microbiology  and  Microanalysis  of  Foods. 
P.  Blakiston's  Son  and  Company.     1920. 

12.  Stitt,  E.  R. — Bacteriology,  Blood  Work  and  Animal  Parasitology. 
P.  Blakiston's  Son  and  Company.     1917. 

13.  Tanner,  Fred  Wilbur. — Bacteriology  and  Mycology  of  Foods. 
John  Wiley  and  Sons.     1919. 

14.  Whipple,  George  Chandler. — The  Microscopy  of  Drinking  Water. 
John  Wiley  and  Sons.     1914. 

15.  Wiley,  H.  W. — Food  and  Their  Aduleration.     P.  Blakiston's  Son 
and  Company.     1907. 

16.  Winton,  Andrew  L. — The  Microscopy  of  Vegetable  Foods.     John 
Wiley  and  Sons.     1906. 


INDEX 


Abrin,  251 
Achorion,  296 
Acid-forming  ferments,  232 
Acromegaly,  288 
Actinomyces,  297 
Addison's  disease,  286,  290 
Adenoids,  287 
Adenology,  277 
Adiposis  dolor  osa,  285,  288 
Adrenal  glands,  290 
Adrenalin,  290 
Aedes  calopus,  319,  387 
Aeration  of  soils,  165 
Aerobic  bacteria,  115 
Aerobioscope,  90 
African  tick  fever,  317 
Agar  gelatin  medium,  75 
Agglutination  test,  378 
Agglutinins,  252 
Aggresins,  253 
Agitation,  324 
Agramonte,  Aristides,  387 
Air  bacteria,  89 
Air,  pure,  323 

Alcoholic  fermentation,  230 
Alinit,  174 
Allergy,  254 
Alumn,  348 
Amboceptor,  245 
Amebae,  316 

Amebo-sphaerocytes,  132 
Ampuls,  362 
Amylase,  226 
Amylopsin,  226 
Amylo-sphaerocytes,  135 
Analysis,  methods  of  205 
Analysis  of  water,  177 
Anaphylaxis,  254 
Anaximander,  8 
Aniline  gentian  violet,  99 
Aniline  water,  98 
Anopheles,  319,  381 


Antagonistic  symbiosis,  146 
Anthrax,  371 
Anti-antitoxin,  252 
Anti-bodies,  251 
Antidiphtheric  serum,  262 

concentrated,  266 
Antigen,  242,  244 
Anti-plague  serum,  268 
Antiseptics,  efficiency  of,  333 

list  of,  332 

respiratory,  331 

tabulation  of,  336 
Antitetanic  serum,  267 
Antitoxin,  252 
Antivenine,  251 
Aphides,  355 
Aristol,  330 

Arnold  steam  sterilizer,  69 
Arrak,  305 
Arrhenius,  24 
Arsenicals,  354 
Arthrospores,  312 
Ascospores,  314 
Asiatic  cholera,  386 
Aspergillus,  297 

flavescens,  388 

fumigatus,  388 

oryzae,  232,  303 

repens,  217 

Association  of  cells,  1 29 
Autodigestion,  223 
Azotobacter,  172,  175 

B 

Bacillus,  acetosum,  234 
acidi  lactici,  185 
anthracis,  371 
Boas-Oppler,  155 
botulinus,  55,  369 
bulgaricus,  201 
butyricus,  185 
Californiensis,  176 
cholerael,  385 


431 


432 


INDEX 


Bacillus,  coli,  253 

diphtherias,  383 

Ellenbachiensis,  172 

fluorescens,  229 

gummosus,  185 

mallei,  371,  373 

oxydans,  234 

Pasteurianum,  234 

pestis,  385 

pneumonias,  379 

soya,  233 

subtilis.  172 

tetani,  373 

tuberculosis,  375 

typhosus,  271,  377 

ureae,  229 

vermiforme,  233 
Bacteria,  in  agriculture,  158 

aerobic,  115 

classification  of,  36,  39 

counting  of,  86,  270 

distribution  of,  60 

growth  of,  83 

isolation  of,  85 

key  to,  48 

morphology  of,  35,  51 

of  air,  89 

of  canned  foods,  91 

of  liquids,  90 

of  milk,  184 

of  mineral  waters,  91 

of  pharmaceuticals,  90 

of  soil,  88,  160 

origin  of,  22 

physiology  of,  35,  56 

protqtrophic,  29 

staining  of,  97 

studying  of,  103 

useful,  158 
Bacterial  chart,  105 

counting,  208 

cultures,  81 

fertilizers,  171 

terminology,  105 
Bacterins,  268 

dose  of,  269 

heterologous,  259 

homologous,  259 

mixed,  259,  276 

preparation  of,  268 

sensitized,  269 


Bacteriology,  400,  412 

history  of,  5 

pharmaceutical,  i 
Bacteriological  technic,  65 
Bacteriological  text  books,  2,  3,  18 
Balantidium,  319 
Balsam  mounts,  426 
Basedow's  disease,  289 
Bathybius,  30 
Beer,  brewing  of,  31 

ginger,  233 
Behring,  14,  238 
Belladonna,  246 
Besredka,  272 
Bichloride  of  mercury,  329 
Billroth,  14 
Biochemistry,  31 
Biologies,  storing  of,  291 
Black  strap,  309 
Blanks,  report,  410,  416 
Blood,  counting,  410 

lakeing  of,  239 

serum,  70 
Borax,  331 

Bordeaux  mixture,  355 
Boric  acid,  331 
Botulism,  369 
Bouillon  nutrient,  70 
Bovine  tuberculosis,  375 
Boveri,«i27 
Broth  medium,  70,  73 
Brown-Sequard,  278 
Brown,  Robert,  120 
Butyric  acid,  185 


Cabinet,  423 

Cachexia  strumipriva,  289 
Cadaver  disinfection,  343 
Calcium  permanganate,  182 
Calmette  test,  375 
Camphor,  332 
Cancer,  384 
Canned  milk,  196 
Capillary  moisture,  164 
Capsule  staining,  102 
Carbolic  acid,  99,  327 
Carbol  fuchin,  99' 
Carbon  disulphide,  354 
Cardan,  8 


INDEX 


433 


Caroll,  James,  387 
Carriers  of  disease,  398 
Castration,  280 
Casts,  408 
Catalysis,  214 
Cat  gut,  sterilization,  361 
Cattle  tick,  317 
Cecropias,  140 
Cell  receptors,  243 
Cells,  association  of,  129 

germatic,  124 

somatic,  124 
Ceni,  389 

Centrifugal  method,  206 
Chalones,  279 
Chart,  bacterial,  105 
Cheese,  191 

Edam,  185 

Chemical  disinfectants. '3 25 
Chicago  canal,  370 
Children,  backward,  283 
Chinatown,  7 
Chlorinated  lime,  330 
Chloroform,  358 
Chloro-sphaerocytes,  135 
Cholera,  386 

Cholera  Commissions,  15 
Chromosomes,  127 
Chromo-sphaerocytes,  134 
Cicatricial  tissue,  247 
Cider,  hard,  301 
Cider  making,  233 
Ciliata,  318 
Circulation,  325 
Citromyces  glaber,  234 

pfefferianus,  234 
Classification  of  bacteria,  36,  39 
Cleaning  of  glassware,  65 
Cleanliness,  323 
Climbing  plants,  141 
Clostridium,  55 
Coefficient,  albumen  coagulating,  333 

phenol,  333 

toxicity,  333 
Cold,  324 

Colloidal  copper,  347 
Colloidal  theory,  30 
Colloids  of  soil,  168 
Colon  bacillus,  95 
Commission,  Cholera,  15 

yellow  fever,  387 

28 


Corpus  luteum,  281 
Communicable  diseases,  369,  395 
Compound  symbiosis,  155 
Copper,  colloidal,  347 

sulphate,  330,  347 
Counting  bacteria,  86 
Counting  chamber,  92 
Counting,  direct,  93 
Counting  plate,  Jeffer's,  117 
Crab,  141 
Crawfish,  141 
Crenothrix,  183 
Creosote,  331 
Cretinism,  285,  289 
Croup,  membranous,  383 
Crime,  284 
Croton,  251 
Cryptococcus,  295 
Cultures,  bacterial,  81 

characteristics  of,  113 

hanging  drop,  116 
Culture  media,  75 

for  mold,  299 

preparation  of,  68 

sterilization  of,  67 
Culture,  stab,  84 

test-tube,  82 
Currie,  358 
Cuscuta,  42 
Cyanides,  354 
Cycas  revoluta,  151 
Cycle,  asexual,  382 

of  Golgi,  382 

human,  382 

life,  126,  127 

mosquito,  382 

of  Ross,  382 

sexual,  382 

Cytoryctes  vaccine  ae,  380 
Cytosis,  156 


Dakin,  321 

Darwin,  121 

Death,  germatic,  127 

somatic,  2 
Deltas,  169 
Dementia  praecox,  284 
Descriptive  chart,  105 
De  Silvestri  test,  378 


434 


INDEX 


Deterioration  of  biologies,  291 

Detre  test,  376 

de  Vries,  121 

Dhobie's  itch,  298 

Diastase,  225 

Diphtheria,  383 

Diplococcus  gonorrheae,  390,  391 

pneumoniae,  379 
Direct  counting,  93 
Discomyces,  297 
Diseases,  369 

Diseases  and  occupation,  374 
Disease,  carriers,  398 

dissemination  of,  397 

immunity  from,  235 

susceptibility  to,  235 
Disinfectants,  321 

action  of,  326 

standardization  of,  322 
Disinfection,  339 

of  buildings,  343 

chemical,  325 

of  clothing,  341 

of  excreta,  340 

of  the  pharmacy,  357 

of  patients,  340 

post  mortem,  343 

public  conveyances,  343 

quarantine,  345 

sick  room,  339,  341 

surgical,  339 

Dissemination  of  disease,  397 
Distribution  of  bacteria,  60 
Doane-Buckley  method,  207 
Dodder,  142 
Dourine,  318 

Ductless  glands,  216,  277,  282 
Dulcin,  353 
Dunham,  21 
Dysentery,  amebic,  317 

• 

E 

Earthquakes,  7 
Echinacea,  246 
Edam  cheese,  185 
Efficiency  value,  333 
Ehrlich,  242,  390 

side  chain  theory,  242 
Electricity,  325 
Electrons,  28 


Ellis,  332 

Embalming  fluid,  343 
Empedocles,  8 
Emulsin,  227 
Endamoeba,  317 
Endocrinology,  277 
Endomyces,  295 
Endospores,  staining  of,  102 
Enzymes,  210 

acid  forming,  234 

classification  of,  218 
Epinephrine,  290 
Erythrasma,  298 
Esmarch  roll-culture,  82 
Eucalyptol,  332 
Europhen,  30 
Eu thymol,  332 
Evolution,  universal,  27 
Exhaustion  theory,  237 
Exhibit  cabinet,  423 

grouping,  429 

microscopic,  422 
Extracts,  glandular,  277     • 


Faeces,  examination,  409 
Fermentation,  210 
Ferments,  210 

classification  of,  218 

acid  forming,  234 
Fertility  of  soils,  167 
Fertilizers,  bacterial,  171 
Fever,  relapsing,  317 
Funfstuck,  154 
Filling  test-tubes,  66 
Finlay,  387 
Filtration,  325 

of  media,  78 
Fixed  virus,  275 
Flagellae,  staining  of,  101 
Flagellata,  317 
Flammarion,  23 
Food  anaphylaxis,  255 
Food  preservatives,  349 
Ford,  181 
Formalin,  328,  358 

generation  of,  344 
Freezine,  352 
Fresh  milk,  186 
Frohlich,  285 


INDEX 


435 


Funnel,  hot  water,  79 
Fusarium,  307 


Galen,  6 

Gametic  fusion,  125 

Gas  formation,  95 

Gas  pressure  regulator,  74 

Gaultherase,  228 

Gay,  272,  279 

Gelatin,  76,  74 

Gemmules,  121 

General  bacterial  method,  208 

Generation,  spontaneous,  n,  22 

Gentian  violet,  99 

Georgics,  159 

Germ  cells,  124 

Germicides,  321 

Gillman,  392 

Ginger  beer,  233 

Glanders,  371 

Glands,  adrenal,  290 

ductless,  216,  277,  282 

extracts,  277 

mammary,  281 

parathyroid,  289 

pineal,  288 

pituitary,  288 

suprarenal,  290 

thymus,  288 

thyroid,  289 

with  ducts,  279 
Glandular  products,  291 
Glassware,  cleaning  of,  65 
Glossina  palpalis,  318 
Glycine  hispidus,  232,  305 
Glycerin  mounts,  427 
Goiter,  286 
Gonoeoccus,  409 
Gonorrhea,  390 
Gordo,  272 
Grades  of  milk,  194 
Graham,  30 
Gram-negative,  101 
Gram-positive,  100 
Gram's  iodine  solution,  100 
Gram  stain,  99 
Grape,  sphaerocytes  of,  135 
Graves'  disease,  289 
Guinea  pigs,  265 


H 

Hsematazoa,  381 
Haffkine,  386 
Halozone,  182 
Hanging  drop  culture,  116 
Hale,  Worth,  334 
Harvey,  120 
Hata,  392 

Heat  sterilization,  324 
Hektoen,  240 
Hemacytometer,  92 
Hemadenology,  277 
Heredity,  123 
Heteroytosis,  157 
Hippocrates,  6 
History  of  bacteriology,  5 
Homunculus,  8,  22 
Hoover,  348 
Hormones,  216,  279 
Horse,  262 
Hot  air  sterilizer,  70 
Hot  water  funnel,  79 
Howard  method,  208 
Humus,  169 
Hydrocyanic  acid,  354 
Hydrogen  dioxide,  331 
Hydrolytic  ferments,  219 
Hydrophobia  vaccine,  275 
Hydrostatic  water,  163 
Hygroscopic  moisture,  164 
Hyphomycetes,  297 
Hypochlorite,  348 


Iceline,  352 
Idants,  121 
Immunity,  235 
Immunization,  238,  357 
Immunology,  235 
Incubator,  73 
Index,  opsonic,  240 
Indigo,  228 
Individualism,  152 
Indol  reaction,  95 
Industrial  bacteria,  158 
Infantilism,  284 
Infectious  diseases,  369 
Influenza,  371 
Infusoria,  318 
Insanity,  284 


436 


INDEX 


Insecticides,  353 
Intoxication  theory,  238 
Introduction,  i 
Invertase,  227 
lodoform,  330 
lodol,  330 
lodo-thyrin,  290 
Iron-forming  bacteria,  58 


Jeffer's  counting  plate,  117 
Jefimoff-Klein  test,  37 
Jenner,  12 
Jequerity  bean,  251 


Kahm  Hefe,  303 
Keffir,  200,  232 
Key,  to  bacteria,  48 
Kircher,  9 
Kitasato,  238 
Kitasato  filter,  83 
Klebs,  13 

Koch,  Robert,  13,  318 
Kolle,  386 
Koumiss,  232 
Kraus,  245 
Kraemer,  347 


Laboratory,  418,  426,  430 
Lactase,  227 
La  grippe,  371 
Laudable  pus,  13,  240,  247 
Lazear,  Jesse,  387 
Leban,  233 
Leeuwenkoek,  9 
Leishman,  240 
Leishmania,  318 
Leprosy  bacillus,  155 
Lesure,  357 
Leucocytosis,  240,  246 
Leuco-sphaerocytes,  133 
Leydenia,  317 
Lichen  symbiosis,  153 
Life,  cycles  of,  126,  127 

malarial,  382 
Light,  325 
Liebig,  211 


Liq.  cres.  comp.,  328 
Lime,  chlorinated,  330 

milk  of,  330 

slaked,  353 
Lipase,  228 
Listerism,  248 
Liver,  279 
Lloyd,  246 
Lobelia,  246 
Lock  jaw,  373 
Loeb,  127 

Loeffler's  methylene  blue,  99 
Loeffler's  serum,  70 
Lombroso,  389 
Longevity,  374 
Lorain  type,  284,  289 
Losophen,  330 
Lymph,  16 
Lupus,  375 
Lysins,  222,  239,  242 

M 

Macallum,  290 
Madura  foot,  298 
Malaria,  381 

control,  383 

plasmodium,  319 
Malassezia,  298 
Mai  de  caderas,  318 
Malta  fever,  191 
Maltase,  226 
Mammary  glands,  281 
Marasmus,  289 
Mayo,  362 
Mazun,  233 
Mead,  Dr.,  8 

Mechanical  disinfectants,  323 
Media,  agar,  74 

agar-gelatin,  75 

beef  extract,  70,  73 

blood  serum,  71 

filtration  of,  76,  78 

gelatin,  74 

LoefHer's,  70 

milk,  71 

peptone,  72 

preparation  of,  75,  78 

sugar-free,  72 

titration  of,  77 
Melicitase,  227 


INDEX 


437 


Mendel,  August,  122 
Menopause,  281 
Menthol,  332 
Merocytes,  382 
Mercury,  bichloride,  329 
Mesophile,  58 
Metacresol,  328 
Metchnikoff,  200,  239 
Methods  of  analysis,  205 
Methyl  alcohol  lamp,  344 
Meyer,  272 
Micellae,  121 
Microanalysis,  401 

urinary,  406 
Microanalysts,  400 
Micrococcus  gonorrheas,  390,  391 
Micrococcus  melitensis,  191 
Microscopic  exhibit,  422 
Microsporoides,  298 
Microsporum,  296 
Migula,  38 

Milk,bacterial  limits  of,  196 
bacteriology  of,  184 
canned,  196 

chemical  examination  of,  199 
fresh,  1 86 
grades  of,  194 
impurities,  193 
of  lime,  330 
medium,  71 
quality  of,  192 
rating  of,  193 
ropy,  1 8 
Mills,  369 

Mills-Reinke  phenomenon,  369 
Moisture,  capillary,  164 

hygroscopic,  164 
Mold,  counting,  208 
Molds,  294 

culture  of,  298 
toxic,  307 

Mongolian  type,  284,  289 
Mordants,  98,  101 
Moro,  test,  376 
Morphology  of  bacteria,  35,  51 
Mother  of  vinegar,  233 
Mounts,  balsam,  426 
glycerin,  427 
smear,  425 
Mucor  mucedo,  294 
species,  307 


Muir's  capsule  staining,  103 
Mutability,  bacterial,  173 
Mutualism,  151 
Mutualistic  symbiosis,  150 
Mutual  parasitism,  147 
Mycodermalaceti,  233 
Mycorhiza,  150 
Myrosin,  228 
Myxedema,  286 

N 

Naegeli,  121 
Naphthalenes,  331 
Nematospora,  311 
Neufeld,  240 
Neutralization,  77 
Nitrogen,  172 
Noguchi,  392 
Normal  solution,  77 
Nosophen,  330 
Nuclein,  246 

Nucleo-sphaerocytes,  134 
Nutrient  bouillon,  70 
Nutricism,  150 


Oberhefe,  295 
Obesity,  284 

Occupation  and  disease,  374 
Ogle,  374 
Ohlmacher,  259 
Oidium  lactis,  191 
Ophthalmia  neonotorum,  390 
Ophthalmo  test,  375 
Oppenheimer,  210 
Opsonic  index,  240 
Opsonins,  240 
Opsonogens,  259 
Origin  of  bacteria,  22 
Origin  of  plasm,  22 
Ornithodoras,  317 
Orthocresol,  328 
Osborne,  33 
Ovaries,  280 
Oxalic  acid,  77 
Oyster,  236 


Pancreas,  279 
Paugenes,  121 
Panspermia,  23 


438 


INDEX 


Papain,  222 

Pappenheim's  stain,  101 
Paracelsus,  8 
Parachymosin,  224 
Paracresol,  328 
Paracytosis,  156 
Paralysis  agitans,  286 
Paramecia,  57,  319 
Parasitism,  146 

mutual,  147 

Parathyroid  glands,  289 
Pandemics,  371 
Paris  green,  354 
Pasteur,  n,  12,  14,  211 
Pasteurization,  189 
Patrocytosis,  156 
Pectase,  224 
Pellagra,  388 
Penicfllium,  296 

glaucum,  298,  388 
Pepsin,  19 

Peptone  medium,  72 
Percutaneans  test,  376 
Permanganate,  potassium,  330 
Pest  exterminators,  203,  353 
Petri  dish,  86 
Petri-dish  culture,  84 
Pfluger,  34 

Phagocytosis,  240,  246 
Pharmaceutical  bacteriology,  i 
Pharmacy,  sterilization,  357 
Pharmacists,  qualifications,  400 
Phenol,  327 

Phenol  coefficient  of,  333 
Phenolphthalein,  77 
Phosphorescens,  58 
Photo-micrographs,  425 
Phycomycetes,  294 
Phylacogens,  276 
Physiology  of  bacteria,  35,  56 
Physiological  resistance,  237 
Phytomer,  123 
Phyton,  123 
Pigmentation,  290 
Pineal  gland,  288 
Pituitary  body,  288 
Placenta,  282 
Plague,  7,  385 
Plague  serum,  268 
Planktom,  203 
Plant  bacteria,  160 


Plant  lice,  355 

Plasmodium  malariae,  319,  381 

Plasm,  origin  of,  22 

Plastids,  123 

Plate  counts,  94,  114 

Plate  cultures,  86 

Platinum,  214 

Platinum  loop,  85 

Platinum  needle,  80 

Plectridium,  55 

Pleons,  21 

Plugging  test-tubes,  64 

Pneumococcus  types,  379 

Pneumonia,  379 

Pollenin,  251 

Polyvalend  bacterins,  276 

Potassium  permanganate,  182,  33 

Potency,  253 

Precipitins,  252 

Precocity,  sexual,  288 

Preparation  of  culture  media,  68,  75,  78 

Prescott-Breed  method,  207 

Preservatives,  food,  349 

Proteases,  219 

Proteolytic  ferments,  219 

Protoplasm,  120 

Prototrophic,  bacteria,  29 

Protozoa  in  disease,  316 

Pseudomonas  radicicola,  104 

Psychrophile,  58 

Pullman  car,  324 

Pus,  definition  of,  247 

formation  of,  247 

laudable,  13,  240,  247 

sanious,  247 

Pyrethrum  fumigation,  320 
Pyroligneous  acid,  353 


Quarantine,  345 
Quinine,  19 


Rabies,  15 

vaccins,  275 
Radium,  in  cancer,  384 
Radium,  28 
Rafter,  183 
Rattlesnake  poisoning,  251 


INDEX 


439 


Ravenel,  275 

Rays,  ultra-violet,  181 

Receptors,  243 

Redi,  Francesco,  10 

Reed,  Walter,  387 

Reichert  thermo-regulator,  74 

Reinke,  369 

Relapsing  fever,  317 

Rennet,  223,  224 

Report  blanks,  410,  416 

Reproduction,  124 

Resistance,  physiological,  237 

Respiratory  antiseptics,  331 

Rhamnase,  228 

Rhizobia,  151 

Rhizobium  leguminosarum,  104 

Rhizobium  mutabile,  104,  171 

Rhizophoda,  316 

Rice  cooker,  72 

Rice  wine,  297 

Rickets,  287 

Ring  burner,  79 

Robin,  251 

Rosenau,  189 

Root  bacteria,  160 

Root  nodules,  171 

Ropy  milk,  185 


Saccharine,  353 
Saccharomyces,  231 

cereviseae,  295 

ellipsoides,  297 

Hanseni,  234 

mycoderma,  234 

pyriformis,  233 

soya,  233 

toxic,  308 
Safety  burner,  79 
Sajous,  283 
Sake,  297 

brewing  of,  301 

filtration  of,  306 
Salamander  poisoning,  251 
Salicylic  acid,  352 
Salvarsan,  390 
Sanibon,  389 
San  Franciso,  7 
Sanious  pus,  247 
Sanitation,  course  in,  417 


Saprophytism,  149 

Sarcina  hamayuchia,  233 

Sarcode,  120 

Scar  tissue,  247 

Schafer,  276 

Schleiden,  120 

Schwann,  9,  12,  211,  120 

Scorpion  poisoning,  251 

Schwarzschild,  24 

Scrofula,  375 

Sedgwick,  183 

Sed wick-Rafter  method,  206 

Sedimentation,  325 

Seminase,  227 

Senility,  premature,  286 

Sensitized  bacterins,  269 

Sera,  258 

Serology,  258 

Serum  anaphylaxis,  254 

Serum,  antidiphtheric,  262 
antitetanic,  267 
concentration,  266 
manufacture  of,  262 
standardization  of,  263 

Sewage  indicators,  179 

Sex  determinant,  127 

Sexual  reproduction,  124 

Sheep  poisoning,  308 

Shering  lamp,  344 

Side  chain  theory,  242 

Simulium  fly,  389 

Skin  reactions,  256 

Sleeping  sickness,  318 

Smallpox,  380 
vaccines,  273 

Smear  preparations,  117 

Smears,  making  of,  425 

Smith's  stain,  101 

Smith,  Theobald,  272 

Smoke  consumer,  323 

Smut,  309 

Sodium  benzoate,  352 

Sodium  hydroxide,  77 

Soil,  aeration,  165 
bacteria,  88,  160 
colloids,  1 68 
fertility,  167 
inoculation,  171 
number  of  bacteria,  87 
temperature,  166 

Sols,  214 


440 


INDEX 


Solution,  normal,  77 

Somatic  cells,  124 

Sour  milk,  preparation  of,  200 

Soya  sauce,  232,  305 

Spirillum,  317 

Spirochaeta,  317 

Spirochaeta  pallida,  389 

Sphaerocytes,  32,  130 

Spleen,  279 

Spontaneous  generation,  u,  22 

Spore,  counting,  208 

staining,  102 
Sporozoites,  382 
Sprays,  355,  356 
Spring  fever,  290 
Sputum,  examination,  409 
Squash,  sphaerocytes  of,  136 
Stab  cultures,  84 
Stable  fly,  236 
Stain,  Gram's,  99 
Staining,  bacteria,  97 

capsule,  102 

flagellae,  101 

spores,  102 
Staphylococcus,  248 
Steam  sterilizer,  Arnold,  69 
Steapsin,  229 
Stegomyia,  319,  387 

by  ultra  rays,  357 

culture  media,  67 

drug  store,  359 
Sterilization,  of  ampuls,  366 

of  catgut,  361 

of  Pharmaceuticals,  358,  366 

of  surgical  supplies,  360 

of  water,  346 

pharmacy,  357 
Sterilizer,  hot  air,  70 
Stewart-Slack  method,  207 
Stich,  357 
Streptococcus,  248 
Streak  cultures,  84 
Sub-cultures,  84,  114 
Sugar-free  medium,  72 
Sulphate  of  copper,  330 
Sulphur,  329,  341 
Sunlight,  325 
Suprarenal  glands,  290 
Surra,  318 
Sucrol,  353 
Symbiology,  119 


Symbiosis,  119 

accidental,  144 
antagonistic,  146 
classification  of,  143 
compound,  155 
contingent,  145 
definition  of,  138 
indifferent,  144 
incipient,  144 
in  lichens,  153 
mutualistic,  150 
origin  of,  138 
phenomena  of,  137 
unclassified,  140 

Syphilis,  389 

Syphilis,  test  for,  392 


Tachycardia,  289 

Tauning  bacteria,  204 

Taurin,  279 

Technic,  bacteriological,  65 

Temperature  of  soils,  166 

Testes,  280 

Tests,  for  disease,  256 

Test-tube  cultures,  82 

inoculation,  82 

filling,  66 

plugging,  64 
Tetanus,  248 
Tetany,  286 
Tete  fever,  317 

Text  books,  bacteriological,  2,  3,  18 
Theory,  colloidal,  30 
Thermal  test,  376 
Thermophile,  58 
Thermo-regulator,  74 
Thymus  gland,  288 
Thyroid  glands,  289 
Thyroiodine,  290 
Thyroidism,  280 
Tissue,  cicatrical,  247 

scar,  247 

transplantation,  122 
Titration,  77 
Tomato,  131 
Tonsils,  287 
Top  yeast,  295 
Torula,  231 
Toxicity  coefficient,  333 


INDEX 


441 


Toxicologists,  400 
Trehalase,  227 

Treponema  pallidium,  317,  389 
Trichophytom,  295 
Trichosporum,  298 
Tricesol,  328 
Troland,  34,  212 
Trypanosoma  Gambiense,  318 
Trypsin,  221 
Tsetse  fly,  318 
Tube  inoculation,  82 
Tuberculins,  272 
Tuberculin  test,  188,  194 
Tuberculosis,  1 88,  375 

urinary  test,  377 
Typhoid  fever,  377 

urinary  test,  378 
Tyrosynase,  230 

U 

Ultra  micro-organisms,  58 
Ultra-violet  rays,  181,  357 
Universal  evolution,  27 
Unterhefe,  296 
Urease,  229,  232 
Urinary  analysis,  406 
Urinary  test,  in  typhoid,  378 

in  tuberculosis,  377 
Ustilago,  309 


Vaccination,  380 

Vaccines,  258 

Vaccines,  hydrophobia,  275 

manufacture  of,  273 

smallpox,  273 
Variola,  380 
Vedder,  181 
Vegetable  rennet,  224 
Vincent's  angina,  318 
Vinegar  making,  233 
Virgil,  159 
Virulency,  253 


Vitalistic  theory,  23 
Von  Pirquet  test,  376 

W 

Washes,  355 
Wassermann  test,  392 
Water  analysis,  177 

bacteriology  of,  176 

copper  treatment,  347 

hydrostatic,  163 

purification  of,  181,  346 
Webster,  Noah,  7 
Weigert's  law,  244 
Weismann,  121 
Wet  blanket  method,  344 
Whipple,  177 
Widal  test,  378 
Williams,  278 
Wilson,  125 
Wine,  rice,  297 
Wolf,  Casper,  121 
Woodworth,  355 
Wright,  1 6,  240 


X-ray,  325 


Yeast,  bottom,  296 

top,  295 
Yeasts,  294 

toxic,  307 

Yellow  fever,  319,  387 
Yersin  serum,  268,  386 
Yoghurt,  232 


Zea,  Mays,  388 
Zygomycetes,  294 
Zymase,  230 
Zymology,  210 


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Sm-2,'37 


Schneider,  A.      40 
Pharmaceutical  bacte 
1920      ology.  2d  ed. 

1938 
JAN  *  y  ^^8 

MAR    9-  1938 
APR  19  1938 


